U.S. patent application number 10/449830 was filed with the patent office on 2004-07-15 for dna sequences from staphylococcus aureus bacteriophage 44ahjd that encode anti-microbial polypeptides.
This patent application is currently assigned to Phagetech, Inc.. Invention is credited to Bergeron, Dominique, DuBow, Michael, Gros, Philippe, Pelletier, Jerry.
Application Number | 20040137516 10/449830 |
Document ID | / |
Family ID | 46299344 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137516 |
Kind Code |
A1 |
Pelletier, Jerry ; et
al. |
July 15, 2004 |
DNA sequences from staphylococcus aureus bacteriophage 44AHJD that
encode anti-microbial polypeptides
Abstract
This invention relates to newly identified polynucleotides and
polypeptides, and their production and uses, as well as their
variants, agonists and antagonists, and their uses. In particular,
the invention relates to specific interaction between the S. aureus
STAAU_R2 related protein or specific regions thereof, and
growth-inhibitory proteins encoded by the S. aureus bacteriophage
genome. The invention relates to the use of these interaction
target sites as the basis of drug screening assays.
Inventors: |
Pelletier, Jerry;
(Baie-D'Urfe, CA) ; Gros, Philippe; (St. Lambert,
CA) ; DuBow, Michael; (Antony, FR) ; Bergeron,
Dominique; (Montreal, CA) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
Phagetech, Inc.
|
Family ID: |
46299344 |
Appl. No.: |
10/449830 |
Filed: |
May 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10449830 |
May 31, 2003 |
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09727892 |
Dec 1, 2000 |
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10449830 |
May 31, 2003 |
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PCT/CA01/01754 |
Nov 30, 2001 |
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60168777 |
Dec 1, 1999 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
A61K 39/00 20130101;
C12N 2795/10022 20130101; C12Q 1/18 20130101; G01N 33/542 20130101;
C07K 14/31 20130101; G01N 33/56938 20130101; C07K 14/005 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53; C12N
001/22 |
Claims
What is claimed is:
1. A method for identifying a compound that is active on a STAAU_R2
polypeptide which comprises the amino acid sequence of SEQ ID NO:
2, a biologically active fragment thereof, or variant thereof,
wherein said amino acid sequence of SEQ ID NO:2, biologically
active fragment thereof, or variant thereof is capable of binding
specifically to a bacteriophage polypeptide sequence, said method
comprising: contacting a STAAU_R2 polypeptide in the presence or
absence of a candidate compound, and detecting a biological
activity of said STAAU_R2 polypeptide, wherein a decrease in the
biological activity thereof in the presence of the candidate
compound relative to the biological activity in the absence thereof
identifies the candidate compound as a compound that is active on
the STAAU_R2 polypeptide.
2. The method of claim 1, wherein said bacteriophage polypeptide
sequence is selected from the group consisting of: a) SEQ ID NO:4;
b) SEQ ID NO:6; c) SEQ ID NO:8; d) SEQ ID NO:10; and e) a fragment
or variant of any one of a) to d), wherein the fragment or variant
thereof maintains its specific binding capability of interacting
with SEQ ID NO:2, fragment or variant thereof.
3. The method of claim 2, wherein said detecting comprises the act
of measuring the binding of said STAAU_R2 polypeptide to said
bacteriophage polypeptide wherein said STAAU_R2 polypeptide or said
bacteriophage polypeptide is directly or indirectly detectably
labeled.
4. The method of any one of claims 1 to 3, wherein said detecting
comprises measurement by FRET.
5. The method of any one of claims 1 to 3, wherein said detecting
comprises measurement of fluorescence polarization changes.
6. The method of any one of claims 1 to 3, wherein said detecting
comprises measurement by surface plasmon resonance.
7. The method of any one of claims 1 to 3, wherein said detecting
comprises a scintillation proximity assay.
8. The method of any one of claims 1 to 3, wherein said detecting
comprises a biosensor assay.
9. The method of any one of claims 1 to 3, wherein said detecting
comprises measurement by phage display.
10. The method of any one of claims 1 to 3, wherein said candidate
compound is selected from the group consisting of a small molecule,
a peptidomimetic compound, and a fragment or derivative of a
bacteriophage inhibitor protein.
11. The method of any one of claims 1 to 3, wherein said candidate
compound is a peptide synthesized by an expression system and
purified, or artificially synthesized.
12. A method for identifying a compound active on one of a STAAU_R2
polypeptide, or on a polypeptide derived from a bacteriophage ORF
which specifically interacts with said STAAU_R2 polypeptide
comprising: contacting a first and a second polypeptide in the
presence or absence of a candidate compound, wherein said first
polypeptide is a STAAU_R2 polypeptide which comprises the amino
acid sequence of SEQ ID NO: 2, fragment, or variant thereof, and
wherein said second polypeptide is a bacteriophage ORF selected
from the group consisting of: a) SEQ ID NO:4; b) SEQ ID NO:6; c)
SEQ ID NO:8; d) SEQ ID NO:10; and e) a fragment or variant of any
one of a) to d), wherein the fragment or variant thereof maintains
its biological activity; and detecting a biological activity of the
first and/or second polypeptide, wherein a decrease in the
biological activity thereof in the presence of the candidate
compound relative to in the absence thereof identifies the
candidate compound as a compound that is active on one of said
STAAU_R2 polypeptide or a polypeptide derived from a
bacteriophage.
13. The method of claim 12, which identifies a compound active on
STAAU_R2.
14. The method of claim 12 or 13, wherein said detecting comprises
the step of measuring the binding of a candidate compound to said
polypeptide, wherein the compound is directly or indirectly
detectably labeled.
15. The method of claim 12 or 13, wherein said detecting comprises
measurement by FRET.
16. The method of claim 12 or 13, wherein said detecting comprises
measurement of fluorescence polarization changes.
17. The method of claim 12 or 13, wherein said detecting comprises
measurement by surface plasmon resonance.
18. The method of claim 12 or 13, wherein said detecting comprises
a scintillation proximity assay.
19. The method of claim 12 or 13, wherein said detecting comprises
a biosensor assay.
20. The method of claim 12 or 13, wherein said detecting comprises
measurement by phage display.
21. The method of claim 12 or 13, wherein said active compound is
selected from the group consisting of a small molecule, a
peptidomimetic compound, and a fragment or derivative of a
bacteriophage inhibitor protein.
22. The method of claim 12 or 13, wherein said active compound is a
peptide synthesized by an expression system and purified, or
artificially synthesized.
23. An agonist or an antagonist of the activity of a STAAU_R2
polypeptide or fragment thereof, or a nucleic acid encoding said
polypeptide or fragment thereof.
24. A method of identifying a compound that is active on a STAAU_R2
polypeptide, said method comprising: contacting a candidate
compound with cells expressing a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, fragment or variant thereof, wherein
said amino acid sequence of SEQ ID NO:2, fragment thereof, or
variant thereof is capable of binding specifically to a
bacteriophage polypeptide sequence, and detecting a STAAU_R2
activity in said cells, wherein a decrease in said activity in said
cells in the presence of said candidate compound is indicative of
an inhibition of STAAU_R2 activity by said compound.
25. A method of making an antibacterial compound, comprising the
steps of: determining whether a candidate compound is active on a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
fragment thereof or variant thereof, or a nucleic acid encoding
said polypeptide, wherein said amino acid sequence of SEQ ID NO:2,
biologically active fragment thereof, or variant thereof is capable
of binding specifically to a bacteriophage polypeptide sequence;
synthesizing or purifying said candidate compound in an amount
sufficient to provide a therapeutic effect when administered to an
organism infected by a bacterium naturally producing said
polypeptide, or nucleic acid encoding same.
26. The method of claim 25, wherein the antibacterial compound is
selected from the group consisting of a small molecule, a
peptidomimetic compound, and a fragment or derivative of a
bacteriophage inhibitor protein.
27. The method of claim 25, wherein the antibacterial compound is a
peptide synthesized by an expression system and purified, or
artificially synthesized.
28. A method for inhibiting a bacterium, comprising contacting the
bacterium with a compound active on a S. aureus polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, or fragment or
variant thereof, or a nucleic acid encoding same.
29. The method of claim 28, wherein said compound is selected from
the group consisting of: a) SEQ ID NO:4; b) SEQ ID NO:6; c) SEQ ID
NO:8; d) SEQ ID NO:10; and e) a fragment or variant of any one of
a) to d), wherein the fragment or variant thereof maintains its
specific binding capability of interacting with SEQ ID NO:2,
fragment or variant thereof.
30. The method of claim 29, wherein said contacting is performed in
vitro.
31. The method of claim 29, wherein said contacting is performed in
vivo in an animal.
32. The method of claim 29, wherein said contacting is performed in
combination with existing antimicrobial agents.
33. The method of claim 29, wherein the antibacterial compound is
selected from the group consisting of a small molecule, a
peptidomimetic compound, and a fragment or derivative of a
bacteriophage inhibitor protein.
34. The method of claim 29, wherein the antibacterial compound is a
peptide synthesized by an expression system and purified, or
artificially synthesized.
35. A method for treating or preventing a bacterial infection in an
animal suffering from an infection, comprising administering to the
animal a therapeutically effective or prophylactic effective amount
of a compound active on a S. aureus polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, fragment or variant thereof,
or a nucleic acid encoding said polypeptide.
36. The method of claim 36, wherein said compound is selected from
the group consisting of: a) SEQ ID NO:4; b) SEQ ID NO:6; c) SEQ ID
NO:8; d) SEQ ID NO:10; and e) a fragment or variant of any one of
a) to d), wherein the fragment or variant thereof maintains its
specific binding capability of interacting with SEQ ID NO:2,
fragment or variant thereof.
37. A method of prophylactic treatment to prevent bacterial
infection comprising contacting an indwelling device with a
compound active on a S. aureus polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, fragment thereof or variant thereof,
before its implantation into a mammal, such contacting being
sufficient to prevent S. aureus infection at the site of
implantation.
38. A method of prophylactic treatment to prevent infection of an
animal by a bacterium comprising administering to the animal a
compound that is active on a S. aureus polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or fragment thereof or variant
thereof, or a gene encoding said polypeptide in an amount
sufficient to reduce adhesion of the bacterium to a tissue surface
of a tissue of the mammal.
39. A composition comprising a STAAU_R2 polypeptide, fragment or
variant thereof, and at least one bacteriophage polypeptide
selected from a bacteriophage 44AHJD ORF 25, Twort ORF 168-encoded
polypeptide, and G1 ORF 240, or a fragment from said bacteriophage
polypeptide.
40. A composition comprising two specifically interacting domains,
wherein said first domain is derived from a STAAU_R2 polypeptide
and said second domain is derived from a polypeptide encoded by a
bacteriophage ORF which specifically interacts with said STAAU_R2
polypeptide.
41. A process for producing a pharmaceutical composition
comprising: a) carrying out a screening assay of the present
invention aimed at identifying a compound that is active on a
STAAU_R2 polypeptide comprising the amino acid sequence of SEQ ID
NO:2, a biologically active fragment thereof, or variant thereof,
wherein said STAAU_R2 polypeptide is capable of binding
specifically to a second polypeptide derived from a bacteriophage
ORF, and wherein the screening assay enables the identification of
a candidate compound as a compound that is active on a STAAU_R2
polypeptide when a biologically activity of said STAAU_R2
polypeptide is measurably different in the presence of said
candidate compound as compared to in the absence thereof; and b)
mixing the compound identified in a) in a pharmaceutically
effective amount with a suitable pharmaceutical carrier, thereby
producing a pharmaceutical composition.
42. Use of one of: a) a STAAU_R2 polypeptide comprising the amino
acid sequence of SEQ ID NO:2, a biologically active fragment
thereof or variant thereof, wherein said STAAU_R2 polypeptide is
capable of binding specifically to a polypeptide derived from a
bacteriophage ORF, b) a composition comprising a pair of
specifically interacting domains comprised of a polypeptide of
STAAU_R2, biologically active fragment thereof or variant thereof
and a polypeptide encoded by a bacteriophage ORF which specifically
interacts with STAAU_R2; or c) an assay mixture comprising a first
polypeptide which comprises the amino acid sequence of SEQ ID NO:2,
biologically active fragment thereof or variant thereof and a
second polypeptide encoded by a bacteriophage ORF which
specifically interact with each other; for the identification of a
compound that is active on a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, biologically active fragment thereof or
variant thereof.
43. Use according to claim 42, wherein said compound active on said
polypeptide is used for the manufacture of an antibacterial agent
or for the manufacture of a medicament for treating or preventing a
bacterial infection.
Description
FIELD OF THE INVENTION
[0001] The invention relates to bacterial genes and proteins that
are implicated in the process of DNA replication and also to
bacteriophage genes and their protein products that interact with
bacterial proteins of DNA replication. More particularly, the
invention relates to compositions and methods involving an
essential Staphylococcus aureus gene and its encoded protein
STAAU_R2. In addition, the invention relates to screening assays to
identify compounds which modulate the level and/or activity of
STAAU_R2 and to such compounds.
BACKGROUND OF THE INVENTION
[0002] The Staphylococci make up a medically important genera of
microbes known to cause several types of diseases in humans. S.
aureus is a Gram positive organism which can be found on the skin
of healthy human hosts and it is responsible for a large number of
bacteremias.
[0003] S. aureus has been successfully treated with the penicillin
derivative Methicillin in the past, but is now becoming
increasingly resistant (MRSA--Methicillin Resistant S. aureus) to
this antibiotic [Harbath et al., (1998) Arch. Intern. Med. 158:
182-189]. For example, S. aureus endocarditis mortality can range
from 26-45%, and combined .beta.-lactam/aminoglycoside therapy is
proving increasingly ineffective in disease eradication [R.o
slashed.der et al., (1999) Arch. Intern. Med. 159: 462-469].
[0004] It is no longer uncommon to isolate S. aureus strains which
are resistant to most of the standard antibiotics, and thus there
is an unmet medical need and demand for new anti-microbial agents,
vaccines, drug screening methods, and diagnostic tests for this
organism. Antibiotics can be grouped into broad classes of
activities against surprisingly few targets within the cell.
Generally, the target molecule is a cellular protein that provides
an essential function. The inhibition of activity of the essential
protein leads either to death of the bacterial cell or to its
inability to proliferate. Critical cellular functions against which
antibiotics are currently in use include cell wall synthesis,
folate and fatty acid metabolism, protein synthesis, and nucleic
acid synthesis.
[0005] A proven approach in the discovery of a new drug, referred
to as target-based drug discovery to distinguish it from cell-based
drug discovery, is to obtain a target protein and to develop in
vitro assays to interfere with the biological function of the
protein. Nucleic acid metabolism is essential for all cells. The
DNA synthesis machinery includes a number of proteins that act in
concert to achieve rapid and highly processive replication of the
chromosome in bacteria [reviewed in Kornberg, A., and Baker, T. A.
1992, DNA Replication, Second edition, New York: W.H. Freeman and
Company, pp. 165-194]. As described below for DNA polymerase III,
biological machines are often comprised of multiprotein complexes.
Coordinated interactions among proteins of the bacterial primosome
and replisome are essential to their efficiency. Thus, any members
of essential multiprotein complexes are hypothetical targets for
drug development. However, the fact that a protein can be
associated with a certain biological function does not necessarily
imply that it represents a suitable target for the development of
new drugs [Drews J. 2000, Science 287:1960-1964]. For instance,
although DNA replication is a well-known and essential process for
bacterial growth, only a relatively small number of DNA replication
proteins are targeted by currently-available antibiotics.
Importantly, screening of compounds for those that inhibit the
function of a target must be preferably rapid and selective.
[0006] There thus remains a need to identify new bacterial targets
and new target domains, and more particularly S. aureus bacterial
targets which could be used to screen for and identify
antibacterial and more particularly anti-S. aureus agents. There
also remains a need to identify new antimicrobial agents, vaccines,
drug screening methods and diagnosis and therapeutic methods.
[0007] The present invention seeks to meet these and other
needs.
[0008] The present description refers to a number of documents, the
content of which is herein incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0009] The present invention relates to new antimicrobial agents,
vaccines, drug screening methods and diagnosis and therapeutic
methods.
[0010] More particularly, the invention relates to proteins which
interact with STAAU_R2 and in particular to bacterial
growth-inhibitory (or inhibitor) bacteriophage gene products that
interacts with the S. aureus STMU.sub.13 R2 polypeptide.
[0011] The invention also relates to a pair of interaction proteins
and parts or fragments thereof. More specifically, the invention
relates to the interacting domains of the S. aureus STAAU_R2
related protein and to proteins which interact with same and block
or inhibit a STAAU_R2 biological activity. In a particular
embodiment, the invention relates to a pair of interacting domains
comprised of that of STAAU_R2 and a polypeptide encoded by a
bacteriophage ORF which specifically interacts with STAAU_R2, such
as the S. aureus bacteriophage 44AHJD ORF 25, Twort ORF168, or G1
ORF 240. In a particularly preferred embodiment of the present
invention, the interaction of these domains and a modulation
thereof forms the basis for screening assays to identify modulators
of STAAU_R2 biological function and more particularly of
antimicrobials.
[0012] The present invention also relates to polynucleotides and
polypeptides of a multiprotein complex believed to be involved in
DNA replication containing STAAU_R2 as a subunit, as well as
variants and portions thereof.
[0013] In another aspect, the invention relates to methods for
using such polypeptides and polynucleotides, including treatment
and diagnosis of microbial diseases, amongst others.
[0014] In a further aspect, the invention relates to methods for
identifying agonists and antagonists using the materials provided
by the invention. In a related aspect, the invention relates to
methods for treating microbial infections and conditions associated
with such infections with the identified agonist or antagonist.
[0015] In a still further aspect, the invention relates to
diagnostic assays for detecting diseases associated with microbial
infections and conditions associated with such infections. In one
embodiment, the diagnostic assay detects STAAU_R2 expression and/or
activity.
[0016] In one particular embodiment of the invention, there is
provided a method of identifying a compound that is active on a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
biologically active fragment, or variant thereof, wherein SEQ ID
NO:2, biologically active fragment, or variant thereof is capable
of binding specifically with a polypeptide comprising the sequence
selected from SEQ ID NOs:4, 6, 8 and 10, a biologically active
fragment thereof, and variant thereof, wherein the fragment or
variant retain their binding capability of specifically interacting
with SEQ ID NO:2 or fragment or variant thereof.
[0017] In one preferred embodiment of the invention, the
identification of a compound active on a STAAU_R2 polypeptide is
provided by a method comprising: contacting a first and a second
polypeptide in the presence or absence of a candidate compound,
wherein the first polypeptide comprises the amino acid sequence of
SEQ ID NO: 2, fragment or variant thereof which specifically bind
to a second polypeptide derived from a bacteriophage ORF which is
capable of binding specifically with SEQ ID NO:2, fragment, or
variant thereof. In one particular embodiment, the second
polypeptide is selected from the group consisting of a phage ORF
(e.g. SEQ ID NO: 4 and 6), a fragment thereof (e.g. SEQ ID NO: 8)
or variant thereof (e.g. SEQ ID NO 10), wherein this second
polypeptide maintains its biological activity; and detecting a
biological activity of the first and/or second polypeptide, wherein
a decrease in the biological activity in the presence thereof
relative to the biological activity in the absence of the candidate
compound identifies the candidate compound as a compound that is
active on a polypeptide comprising the amino acid sequence of SEQ
ID NO:2, fragment or variant thereof.
[0018] In one particular embodiment, the biological activity is the
binding of the first and second polypeptides to each other, the
method comprising: contacting an assay mixture comprising a) a
first polypeptide which comprises the amino acid sequence of SEQ ID
NO:2 or a biologically active fragment thereof, or variant thereof,
and b) a second polypeptide selected from the group consisting of
SEQ ID NO:4, 6, 8, 10, a fragment thereof, and a variant thereof;
with a test compound; measuring the binding of the first and the
second polypeptides in the presence of the candidate compound
relative to the binding in the absence thereof and; determining the
ability of the candidate compound to interact with a STAAU_R2
polypeptide or variant thereof, wherein a decrease in the binding
of the first and the second polypeptides in the presence of the
candidate compound that interacts with STAAU_R2, relative to the
binding in the absence of the candidate compound, identifies the
candidate compound as a compound that is active on a STAAU_R2
polypeptide.
[0019] In another embodiment of the present invention, there is
provided a process for producing a pharmaceutical composition
comprising: a) carrying out a screening assay of the present
invention aimed at identifying a compound that is active on a
STAAU_R2 polypeptide comprising the amino acid sequence of SEQ ID
NO:2, biologically active fragment, or variant thereof, wherein the
STAAU_R2 polypeptide is capable of binding specifically with a
second polypeptide derived from a bacteriophage ORF, and wherein
the screening assay enables the identification of a candidate
compound as a compound that is active on a STAAU_R2 polypeptide;
and b) mixing the compound identified in a) to a suitable
pharmaceutical carrier. In a further embodiment of this process of
producing a pharmaceutical composition, the process further
includes a scaling-up of the preparation for isolating of the
identified compound active on the STAAU_R2 polypeptide. In yet
another embodiment of this process of producing a pharmaceutical
composition, the pharmaceutical composition prepared comprises a
derivative or homolog of the compound identified in a).
[0020] In yet another embodiment of the present invention, there is
provided one of a use of a) a STAAU_R2 polypeptide comprising the
amino acid sequence of SEQ ID NO:2, a biologically active fragment
thereof or variant thereof, wherein SEQ ID NO:2, biologically
active fragment thereof or variant thereof (e.g. the STAAU_R2
polypeptide) is capable of binding specifically to a polypeptide
derived from a bacteriophage ORF, b) a composition comprising a
pair of specifically interacting domains comprised of a polypeptide
of STAAU_R2, biologically active fragment thereof or variant
thereof and a polypeptide encoded by a bacteriophage ORF which
specifically interacts with STAAU_R2; or c) an assay mixture
comprising a first polypeptide which comprises the amino acid
sequence of SEQ ID NO:2, biologically active fragment thereof or
variant thereof and a second polypeptide encoded by a bacteriophage
ORF which specifically interact with each other; for the
identification of a compound that is active on a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, biologically
active fragment thereof or variant thereof. In a particularly
preferred embodiment of the present invention, the bacteriophage
polypeptide sequence is selected from the group consisting of SEQ
NOs: 4, 6, 8, 10, a fragment thereof and a variant thereof, wherein
the fragment thereof or variant thereof retain their specific
binding capability to STAAU_R2 polypeptide.
[0021] In one embodiment, the step of detecting comprises the step
of measuring the binding of the first and second proteins, wherein
the first or the second protein is directly or indirectly
detectably labeled.
[0022] In different embodiments, the step of detecting comprises,
but is no limited to, measurement by the method selected from the
group consisting of fluorescence resonance energy transfer,
fluorescence polarization changes, measurement by surface plasmon
resonance, a scintillation proximity assay, a biosensor assay, and
phage display.
[0023] In one embodiment, a library of compounds is used.
Non-limiting examples of candidate compounds include a small
molecule, a peptidomimetic compound, a peptide, and a fragment or
derivative of a bacteriophage inhibitor protein.
[0024] In one embodiment, the candidate compound is a peptide
synthesized by expression systems and purified, or artificially
synthesized.
[0025] The invention also encompasses a method of identifying an
antimicrobial agent comprising determining whether a test compound
is active on a S. aureus polypeptide, namely STAAU_R2 as set forth
in SEQ ID NO: 2, or parts thereof.
[0026] In a further embodiment, identifying a compound active on a
STAAU_R2 polypeptide is provided by a method which comprises:
contacting a candidate compound with a polypeptide comprising the
amino acid sequence of SEQ ID NO: 2; a fragment thereof or a
variant thereof, the fragment or variant retaining its biological
activity (e.g. it specifically binds one of the sequences selected
from SEQ ID NOs: 4, 6, 8, 10, fragment thereof, or variant thereof
(SEQ ID NO: 10)), and detecting binding of the candidate compound
thereto, wherein detection of binding is indicative that the
compound is active on the polypeptide.
[0027] In different embodiments, the step of detecting includes
measuring the binding of a candidate compound to the polypeptide,
wherein the compound is directly or indirectly detectably labeled,
by a method comprising, but not limited to, fluorescence resonance
energy transfer, fluorescence polarization changes, measurement by
surface plasmon resonance, scintillation proximity assay, biosensor
assay, and phage display.
[0028] In one embodiment, a library of compounds is used.
Non-limiting examples of candidate compound include a small
molecule, a peptidomimetic compound, a peptide, and a fragment or
derivative of a bacteriophage inhibitor protein.
[0029] In one embodiment, the candidate compound is a peptide
synthesized by expression systems and purified, or artificially
synthesized.
[0030] The invention further encompasses a method of identifying a
compound that is active on a STAAU_R2 polypeptide, comprising the
steps of contacting a candidate compound (or library thereof) with
cells expressing a polypeptide comprising SEQ ID NO: 2; and
detecting STAAU_R2 activity in the cells, wherein a decrease in
activity relative to STAAU_R2 activity in cells not contacted with
a candidate compound is indicative of inhibition of STAAU_R2
activity. The invention also encompasses such a method but using a
fragment or variant of SEQ ID NO:2.
[0031] Of course, the invention further encompasses methods of
identifying a compound that modulates the activity of a STAAU_R2
polypeptide, wherein a compound increasing the activity relative to
STAAU_R2 activity in cells not contacted with the candidate
compound, is selected as a compound which is a stimulator of
STAAU_R2 activity.
[0032] In a preferred embodiment, the step of detecting comprises a
method of measuring the ability of a candidate, test compounds, or
agents to stimulate or preferably to inhibit a STAAU_R2 molecule's
ability to modulate DNA synthesis (such assays are described in
more detail hereinbelow).
[0033] The invention further encompasses a method of identifying a
compound that is active on a STAAU_R2 polypeptide, comprising the
steps of contacting a candidate compound (or library thereof) in a
cell-free assay, with a STAAU_R2 protein or biologically active
portion thereof, either naturally occurring or recombinant in
origin; and detecting STAAU_R2 activity, wherein a decrease in
activity relative to STAAU_R2 activity in cell-free assay not
contacted with a candidate compound is indicative of inhibition of
STAAU_R2 activity.
[0034] In a preferred embodiment, the step of detecting comprises a
method of measuring the ability of a candidate compound, test
compounds, or agents to stimulate, or preferably to inhibit a
STAAU_R2 molecule's ability to modulate DNA synthesis (such assays
are described in more detail hereinbelow).
[0035] The invention further encompasses an agonist or an
antagonist of the activity of a STAAU_R2 polypeptide or a nucleic
acid or gene encoding the polypeptide.
[0036] The assays described herein may be used as initial or
primary screens to detect promising lead compounds for further
development. The same assays may also be used in a secondary
screening assay to measure the activity of candidate compounds on a
STAAU_R2 polypeptide. Often, lead compounds will be further
assessed in additional, different screens. This invention also
includes secondary STAAU_R2 screens which may involve biological
assays utilizing S. aureus strains or other suitable bacteria.
[0037] Tertiary screens may involve the study of the effect of the
agent in an animal. Accordingly, it is within the scope of this
invention to further use an agent identified as described herein in
an appropriate animal model. For example, an test compound
identified as described herein (e.g., a STAAU_R2 inhibiting agent,
an antisense STAAU_R2 nucleic acid molecule, a STAAU_R2-specific
antibody, or a STAAU_R2-binding partner) can be used in an animal
model to determine the efficacy, toxicity, or side effects of
treatment with such an agent. Alternatively, an agent identified as
described herein can be used in an animal model to determine the
mechanism of action of such an agent. Furthermore, this invention
pertains to uses of novel agents identified by the above-described
screening assays for treatment (e.g. bacterial infections), as
described herein.
[0038] The invention further encompasses a method of making an
antibacterial compound, comprising the steps of: a) determining
whether a candidate compound is active on a polypeptide comprising
the amino acid sequence of SEQ ID NO: 2, fragment or variant
thereof, or a gene encoding the polypeptide; and b) synthesizing or
purifying the candidate compound in an amount sufficient to provide
a therapeutic effect when administered to an organism infected by a
bacterium naturally producing a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, fragment or variant thereof.
[0039] The invention further encompasses a method for inhibiting a
bacterium, comprising contacting the bacterium with a compound
active on a polypeptide comprising the amino acid sequence of SEQ
ID NO: 2, fragment or variant thereof, or a nucleic acid encoding
the polypeptide.
[0040] In one embodiment, the step of contacting is performed in
vitro.
[0041] In another embodiment, the step of contacting is performed
in vivo in an animal.
[0042] In another embodiment, bacterium is contacted with the
active compound in combination with existing antimicrobial agents.
Thus, the invention also relates to antimicrobial compositions
comprising a compound of the present invention in combination with
an existing antimicrobial agent. Of course, more than one active
compound of the present invention could be combined with or without
existing antimicrobial agent(s).
[0043] The invention further encompasses a method for treating or
preventing a bacterial infection in an animal suffering from an
infection or susceptible of suffering from same, comprising
administering thereto a therapeutically effective amount of a
compound active on a polypeptide comprising the amino acid sequence
of SEQ ID NO: 2, variant or fragment thereof, or nucleic acid
sequence encoding same. The animal is preferably, but not
necessarily a mammal, and more preferably a human. In one
embodiment, the active compound is administred to the animal in
combination with existing antimicrobial agents. Thus, the invention
also relates to antimicrobial compositions comprising a compound of
the present invention in combination with an existing antimicrobial
agent.
[0044] The invention further encompasses a method of prophylactic
treatment to prevent bacterial infection comprising contacting an
indwelling device with a compound active on a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, variant or
fragment thereof before its implantation into a mammal, such
contacting being sufficient to prevent S. aureus infection at the
site of implantation.
[0045] The invention further encompasses a method of prophylactic
treatment to prevent infection of an animal by a bacterium
comprising administering to the animal a prophylactically effective
amount of a compound that is active on a polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, variant or fragment thereof or
a gene encoding the polypeptide in an amount sufficient to prevent
infection of the animal. In a particular embodiment, the
prophylactically effective amount reduces adhesion of the bacterium
to a tissue surface of the mammal.
[0046] The invention further encompasses a method of diagnosing in
an animal an infection with S. aureus, comprising: determining the
presence in the animal of a polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, part thereof, variant thereof, fragment
thereof, epitope thereof or nucleic acid encoding same. Preferably
the polypeptide is capable of specifically interacting with at
least one of 44AHJD ORF 25, Twort ORF168 or G1 ORF 240. Preferably,
the animal is a human.
[0047] In one embodiment, the determining step comprises contacting
a biological sample of the animal or individual with an antibody
specific for an epitope present on a polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, variant or fragment
thereof.
[0048] The invention further encompasses a method of diagnosing in
an animal or individual an infection with Staphylococcus aureus,
comprising determining the presence in the animal or individual of
a nucleic acid sequence encoding a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, variant or fragment thereof, wherein
the polypeptide is capable of specifically interaction with at
least one of 44AHJD ORF 25, Twort ORF168 or G1 ORF 240.
[0049] In one embodiment, the determining step comprises contacting
a nucleic acid sample of the animal or individual with an isolated,
purified or enriched nucleic acid probe of at least 15 nucleotides
in length that hybridizes under stringent hybridization conditions
with the sequence of SEQ ID NO: 1, or the complement thereof.
[0050] The invention further encompasses a composition comprising
two polypeptides, a bacteriophage-encoded polypeptide and a S.
aureus STAAU_R2 polypeptide corresponding to SEQ ID NO: 2. In
another embodiment, the invention encompasses a composition
comprising two interacting polypeptides derived from a
bacteriophage encoded polypeptide and a S. aureus STAAU_R2
polypeptide. As such, the invention encompasses a composition
comprising two nucleic acid sequences encoding these interacting
polypeptides.
[0051] Further features and advantages of the invention will become
more fully apparent in the following description of the embodiments
and drawings thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] Having thus generally described the invention, reference
will now be made to the accompanying drawings, showing by way of
illustration a preferred embodiment thereof, and in which:
[0053] FIG. 1 shows the nucleotide (SEQ ID NO: 1) and amino acid
(SEQ ID NO: 2) sequences of S. aureus STAAU_R2.
[0054] FIG. 2 shows the nucleotide and the amino acid sequences of
S. aureus bacteriophage 44AHJD ORF 25, Twort ORF168, a 36 amino
acids fragment derived from Twort ORF 168 and G1 ORF 240.
[0055] FIG. 3 shows the bacterial inhibitory potential of
bacteriophage 44AHJD ORF 25 or Twort ORF168 and the expression
vector used to induce their expression in S. aureus. A) Schematic
diagram of arsenite-inducible expression vector (e.g. for pT/ORF
and pTM/ORF) used to induce expression of 44AHJD ORF 25 or Twort
ORF168 in S. aureus cells; B) Results of an assay for inhibitory
potential of 44AHJD ORF 25 or Twort ORF168 when expressed in S.
aureus grown in liquid medium followed by plating on semi-solid
medium either containing or not containing the antibiotic (30 ug/ml
of kanamycin) necessary to maintain the selective pressure for the
plasmid.
[0056] FIG. 4 depicts the results from affinity chromatography
using GST and GST/44AHJD ORF 25 as ligands with a S. aureus extract
prepared by French pressure cell lysis and sonication. Eluates from
affinity columns containing the GST and GST/ORF25 ligands at 0,
0.1, 0.5, 1.0, and 2.0 mg/ml resin were resolved by SDS-12.5% PAGE.
Proteins were visualized by silver staining. Micro-columns were
sequentially eluted with 100 mM ACB containing 0.1% Triton X-100, 1
M NaCl ACB, and 1% SDS (SDS-PAGE only shows for 1% SDS). Each
molecular weight marker is approximately 100 ng. The lanes labeled
ACB indicate eluates from a 2.0 mg/ml ligand column loaded only
with ACB buffer containing 75 mM NaCl. The arrow designated PT 48
indicates protein specifically interacting with 44AHJD ORF 25. Band
corresponding to PT 48 was excised for protein identification.
[0057] FIG. 5 shows results of a tryptic peptide mass spectrum of
the PT 48 protein that interacted with 44AHJD ORF 25 and that was
eluted with 1% SDS. The control band (designated PT48C in FIG. 4)
excised from the 48 kDA region did not contain PT 48.
[0058] FIG. 6 shows the identification of PT 48 (herein STAAU_R2)
as S. aureus DNA-directed DNA polymerase III beta subunit protein
from the Genbank database (accession number: GI:15922992).
[0059] FIG. 7 shows affinity chromatography using GST/Twort ORF 168
(A) or GST (B) as ligands with a 5.0 mg/ml S. aureus extract.
Eluates from affinity columns containing the ligands at 0, 0.1,
0.5, 1.0, and 2.0 mg/ml resin were resolved by 14% SDS-PAGE and the
gel was stained with silver nitrate. Micro-columns were
sequentially eluted with 100 mM ACB containing 0.1% Triton X-100
(SDS-PAGE not shown), 1 M NaCl ACB, and 1% SDS. Each molecular
weight marker is approximately 200 ng. The lanes labeled ACB
indicate eluates from a 2.0 mg/ml ligand column loaded only with
ACB buffer containing 100 mM NaCl. The arrow designated PT50
indicates protein specifically interacting with Twort ORF 168. Band
corresponding to PT50 was excised for protein identification.
[0060] FIG. 8 shows the tryptic peptide mass spectrum analysis of
the PT50 protein interacting with Twort ORF 168. The gel slice
containing PT50 contained one protein.
[0061] FIG. 9 shows the identification of PT50 (herein STAAU_R2) as
S. aureus DNA-directed DNA polymerase III beta subunit protein from
the Genbank database (accession number: GI:15922992).
[0062] FIG. 10 shows schematic representations of A) the procedure
for cloning S. aureus STAAU_R2 in the yeast expression vector
pGADT7 (pGADSTAAU_R2); B) the procedure for cloning phage Twort ORF
168 in the yeast expression vector pGBKT7 (pGBK TwortORF168); and
C) the yeast two-hybrid system in three stylized cells expressing
either GADSTAAU_R2 (top panel), Twort ORF 168 (middle panel), or
both GADSTAAU_R2 and Twort ORF 168 (bottom panel).
[0063] FIG. 11 shows the results of yeast two hybrid analyses
designed to test the interaction of S. aureus STAAU_R2 comprising
the amino acid of SEQ ID NO: 2 and Twort ORF 168. A) and B). Yeasts
were co-transformed with pairs of vectors as indicated above each
pair of photographs of Petri plates. Co-transformants were plated
in parallel on yeast synthetic medium (SD) supplemented with amino
acid drop-out lacking tryptophan and leucine (TL minus) and on SD
supplemented with amino acid drop-out lacking tryptophan,
histidine, adenine and leucine (THAL minus). Co-transformants
harboring the Twort ORF 168 polypeptide only grew on selective THAL
minus media in the presence of STAAU_R2 (top pairs of petri
plates). Co-transformation of these polypeptides with control
vectors harboring non-interacting proteins (pGBKLaminC or pGADT7-T)
does not result in growth on THAL minus medium. C) Results of the
luminescent .beta.-galactosidase enzymatic assays with protein
extracts from the indicated co-transformants.
[0064] FIG. 12 shows optimal global and local alignments of Twort
ORF168 (SEQ ID NO:6) and the STAAU_R2 interaction domain of Twort
ORF168 (SEQ ID NO:8) with that of G1 ORF 240 (SEQ ID NO: 10).
[0065] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference
to the accompanying drawing which is exemplary and should not be
interpreted as limiting the scope of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0066] The invention relates to the discovery of an essential gene
and its encoded polypeptide in S. aureus and portions thereof
useful for example in screening, diagnostics, and therapeutics.
More specifically, the invention also relates to S. aureus STAAU_R2
polypeptides and polynucleotides as described in greater detail
below, and to a pair of polynucleotides encoding a pair of
interacting polypeptides, to the pair of polypeptides themselves,
or interacting domains thereof. In a particular embodiment, the
pair includes a S. aureus STAAU_R2 polypeptide or interacting
domain thereof and a) a 44AHDJ ORF 25 or interacting domain
thereof; b) a Twort ORF168 or interacting domain thereof; or c) a
G1 ORF 240 or interacting domain thereof. In one embodiment, the
invention relates to STAAU_R2 having the nucleotide and amino acid
sequences disclosed as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The sequences presented as SEQ ID NOs: 1 and 2 represent an
exemplification of the invention, since those of ordinary skill
will recognize that such sequences can be usefully employed in
polynucleotides in general, including ribopolynucleotides.
[0067] The methodology of two previous inventions (U.S. Provisional
Patent Application 60/110,992, filed Dec. 3, 1998, and PCT
International Application WO1999/IB99/02040, filed Dec. 3, 1999)
has been used to identify and characterize essential polynucleotide
and polypeptide sequences from S. aureus.
[0068] Thus, in a particular embodiment of the present invention,
the present invention provides polynucleotide and polypeptide
sequences isolated from S. aureus that can be used in a drug
screening assay to identify compounds with anti-microbial activity.
The polynucleotide and polypeptide sequences can be isolated using
a method similar to those described herein, or using another
method. In addition, such polynucleotide and polypeptide-sequences
can be chemically synthesized. The identification of these S.
aureus sequences as targets for three different bacteriophages
validates the approach of the present invention to identify
bacterial targets and also validates STAAU_R2 as a key target for
antibacterial drug development as well as diagnosis and treatment
methods based thereon.
[0069] Definitions
[0070] In order to provide a clear and consistent understanding of
terms used in the present description, a number of definitions are
provided hereinbelow.
[0071] The terminology "active on", with reference to a particular
cellular target, such as the product of a particular gene, means
that the target is an important part of a cellular pathway which
includes that target and that an agent or compound acts on that
pathway. Thus, in some cases the agent or compound may act on a
component upstream or downstream of the stated target (i.e.
indirectly on the target), including a regulator of that pathway or
a component of that pathway. In general, an antibacterial agent is
active on an essential cellular function, often on a product of an
essential gene (i.e. directly on the target).
[0072] The terminology "active on" also refers to a measurable
effect of the compound on the target it is active on (as compared
to the activity of the target in the absence of the compound). The
activity referred thereto is any one of a biological activity of
one of the polypeptides of the present invention.
[0073] As used herein, the terms "inhibit", "inhibition",
"inhibitory", and "inhibitor" all refer to a function of reducing a
biological activity or function. Such reduction in activity or
function can, for example, be in connection with a cellular
component (e.g., an enzyme), or in connection with a cellular
process (e.g., synthesis of a particular protein), or in connection
with an overall process of a cell (e.g., cell growth). In reference
to cell growth, the inhibitory effects may be bacteriocidal
(killing of bacterial cells) or bacteriostatic (i.e.--stopping or
at least slowing bacterial cell growth). The latter slows or
prevents cell growth such that fewer cells of the strain are
produced relative to uninhibited cells over a given time period.
From a molecular standpoint, such inhibition may equate with a
reduction in the level of, or elimination of, the transcription
and/or translation and/or stability of a specific bacterial
target(s), and/or reduction or elimination of activity of a
particular target biomolecule.
[0074] As used herein, the term "STAAU_R2 polypeptide" "dnaN
polypeptide" refers to a polypeptide encompassing S. aureus
STAAU_R2 (SEQ ID NO: 2), variant thereof or an active domain of S.
aureus STAAU_R2. As used herein, the term "active domain of S.
aureus STAAU_R2", "biologically active polypeptide of STAAU_R2" or
the like refers to a polypeptide fragment or portion of S. aureus
STAAU_R2 that retains an activity of S. aureus STAAU_R2. The term
"STAAU_R2 polypeptide" is meant to encompass S. aureus STAAU_R2 or
an active domain of S. aureus STAAU_R2 that is fused to another,
non-STAAU_R2 polypeptide sequence.
[0075] "STAAU_R2 activity" "polypeptide comprising the amino acid
sequence SEQ ID NO: 2 activity" "dnaN polypeptide activity" or
"biological activity" of STAAU_R2 or other polypeptides of the
present invention is defined as a detectable biological activity of
a gene, nucleic acid sequence, protein or polypeptide of the
present invention. This includes any physiological function
attributable to the specific biological activity of STAAU_R2, or
phage ORF of the present invention. This includes measurement of
the DNA synthesis activities of STAAU_R2 in cells or in vitro.
Non-limiting examples of the biological activities may be made
directly or indirectly. STAAU_R2 biological activity, for example,
is not limited, however, to its function in DNA synthesis.
Biological activities may also include simple binding to other
factors (polypeptides or otherwise), including compounds,
substrates, and of course interacting proteins. Thus, for STAAU_R2,
biological activity includes any standard biochemical measurement
of STAAU_R2 such as conformational changes, phosphorylation status
or any other feature of the protein that can be measured with
techniques known in the art. STAAU_R2 biological activity also
includes activities related to STAAU_R2 gene transcription or
translation, or any biological activities of such transcripts or
translation products. The instant invention is also concerned with
STAAU_R2 interaction with an inhibitory polypeptide of the present
invention, biological activity of STAAU_R2 also includes assays
which monitor binding and other biochemical measurements of these
polypeptides. Furthermore, for certainty, the terminology
"biological activity" also includes measurements based on the
interaction of domains of interacting proteins of the present
invention (i.e. the phage ORFs or domains thereof). Non-limiting
examples of "biological activity" include one or more of the
following:
[0076] i) Binding to bacterial growth inhibitory ORFs derived from
bacteriophage including 44AHDJ ORF 25, Twort ORF168, G1 ORF 240
polypeptides or part thereof.
[0077] Determining the binding between polypeptides of the present
invention can be accomplished by one of the methods described below
or known in the art for determining direct binding. While it might
be advantageous in certain embodiments of the present invention to
provide a binding assay which is amenable to automation and more
particularly to high-throughput, the present invention is not so
limited. The binding or physical interaction of a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, provided
herein, or fragment thereof to bacteriophage protein 44AHDJ ORF 25,
Twort ORF168, or G1 ORF 240 or portion thereof (e.g. SEQ ID NO: 8).
The interaction or binding of a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2 and a binding portion of
bacteriophage 44AHDJ ORF 25, Twort ORF168, G1 ORF 240 may be
between isolated polypeptides consisting essentially of the
sequence necessary for binding, or, alternatively, the respective
polypeptide sequence may be comprised within a larger
polypeptide.
[0078] A number of non-limiting methods, useful in the invention,
to measure the binding of bacteriophage 44AHDJ ORF 25, Twort
ORF168, G1 ORF 240 to a polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, or fragment thereof are described below.
Binding can be measured by coupling one molecule to a surface or
support such as a membrane, a microtiter plate well, or a
microarray chip, and monitoring binding of a second molecule by any
number of means including optical spectroscopy, fluorometry, and
radioactive label detection.
[0079] For example, Fluorescence Resonance Energy Transfer (FRET),
in which the close proximity of two fluorophores, whether intrinsic
to, as in the case of a naturally-fluorescent amino acid residue
such as tryptophan, or either covalently or non-covalently bound to
a separate molecule, causes the emission spectrum of one
fluorophore to overlap with the excitation spectrum of the second,
and thus dual fluorescence following excitation of only one
fluorophore is indicative of binding. For example, Fluorescence
Resonance Energy Transfer (FRET), in which the close proximity of
two fluorophores, whether intrinsic to, as in the case of a
naturally-fluorescent amino acid residue such as tryptophan, or
either covalently or non-covalently bound to a separate molecule,
causes the emission spectrum of one fluorophore to overlap with the
excitation spectrum of the second, and thus dual fluorescence
following excitation of only one fluorophore is indicative of
binding. An additional assay useful in the present invention is
fluorescence polarization, in which the quantifiable polarization
value for a given fluorescently-tagged molecule is altered upon
binding to a second molecule. Surface plasmon resonance assays can
be used as a quantitative method to measure binding between two
molecules by the change in mass near an immobilized sensor caused
by the binding of one protein from the aqueous phase to a second
immobilized on the sensor. A scintillation proximity assay can also
be used to measure binding of a polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, and fragment thereof and a
bacteriophage ORF or fragment thereof in which binding in the
proximity to a scintillant converts radioactive particles into a
photon signal that is detected by a scintillation counter or other
detector. Additionally, binding can be evaluated by a Bio Sensor
assay, which is based on the ability of the sensor to register
changes in admittance induced by ion-channel modulation following
binding. Phage display is also a powerful quantitative assay to
measure protein:protein interaction using colourimetric ELISA
(enzyme-linked immunosorbent assay).
[0080] ii) Stimulation of DNA Synthesis
[0081] The biological activity also relates to DNA synthesis
stimulation of a polypeptide having the S. aureus STAAU_R2 sequence
provided herein, a fragment or variant thereof, or a protein
comprising a S. aureus STAAU_R2 polypeptide that directly interacts
with bacteriophage protein 44AHJD ORF 25, Twort ORF168, G1 ORF 240,
or a STAAU_R2-binding fragment of the 44AHJD ORF 25, Twort ORF168,
G1 ORF 240 proteins or variant thereof.
[0082] A number of methods, useful in the invention, to measure the
DNA synthesis stimulation of a polypeptide comprising the amino
acid sequence of STAAU_R2 are described below. The level of DNA
synthesis can be evaluated for example by the measurement of
radiolabeled nucleotides incorporation into DNA of S. aureus
cells.
[0083] The rate and processivity of DNA synthesis could also be
measured by using soluble in vitro systems based on the use of a
variety of different synthetic DNA substrates including
single-stranded (ss) DNA, double-stranded (ds) DNA, either linear
or circular. In one embodiment, the replication assay involves
crude or partially purified cellular proteins extracts or
recombinantly produced proteins. In another embodiment, the
reconstituted protein assays involves partially purified or pure
forms of native proteins or fusion proteins or fragments
thereof.
[0084] iii) Loading onto DNA
[0085] The DNA loading of a polypeptide having the S. aureus
STAAU_R2 sequence provided herein, a fragment or analog thereof or
a protein comprising a S. aureus STAAU_R2 polypeptide that directly
interacts with one of bacteriophage 44AHJD ORF 25, Twort ORF168, G1
ORF 240 proteins, or a STAAU_R2-binding fragment of the 44AHJD ORF
25, Twort ORF168, G1 ORF 240 proteins or variant thereof can also
be monitored. In one embodiment, an in vitro reconstituted assay
involves the measurement of .sup.32P-labeled or
fluorescently-labeled STAAU_R2 assembly onto a circular DNA
substrate.
[0086] As used herein, the term "polynucleotide encoding a
polypeptide" or equivalent language encompasses polynucleotides
that include a sequence encoding a polypeptide of the invention,
particularly a bacterial polypeptide and more particularly a
polypeptide of S. aureus STAAU_R2 protein having an amino acid
sequence set out in FIG. 1, SEQ ID NO: 2. The term also encompasses
polynucleotides that include a single continuous region or
discontinuous regions encoding the polypeptide (for example,
polynucleotides interrupted by integrated phage, an integrated
insertion sequence, an integrated vector sequence, an integrated
transposon sequence, or otherwise altered due to RNA editing or
genomic DNA reorganization) together with additional regions that
also may contain coding and/or non-coding sequences.
[0087] As used herein, the term "STAAU_R2 gene" "DnaN gene" is
meant to encompass a polynucleotide encoding a S. aureus STAAU_R2
polypeptide. Any additional nucleotide sequences necessary to
direct transcription of RNA encoding a S. aureus STAAU_R2
polypeptide, either in a cell or in vitro, will be termed
"regulatory sequences", which include but are not limited to
transcriptional promoters and enhancers, and transcription
terminators.
[0088] As used herein, the term "ORF 25" or "phage 44AHJD ORF 25"
or "44AHJD ORF 25" encompasses a polynucleotide having the sequence
provided in FIG. 2 (SEQ ID NO: 3), which encodes a gene product
known as the 44AHJD ORF 25 gene product.
[0089] As used herein, the term "ORF 168" or "phage Twort ORF 168"
or "Twort ORF 168" encompasses a polynucleotide having the sequence
provided in FIG. 2 (SEQ ID NO: 5), which encodes a gene product
known as the Twort ORF 168 gene product.
[0090] As used herein, the term "ORF 240" or "phage G1 ORF 240"
encompasses a polynucleotide having the sequence provided in FIG. 2
(SEQ ID NO: 9). which encodes a gene product known as the G1 ORF
240 gene product.
[0091] As used herein, the term "polynucleotide(s)" generally
refers to any polyribonucleotide or poly-deoxyribonucleotide, which
may be unmodified RNA or DNA or modified RNA or DNA.
"Polynucleotide(s)" include, without limitation, single- and
double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions or single-, double- and triple-stranded
regions, single- and double-stranded RNA, and RNA that is mixture
of single- and double-stranded regions, hybrid molecules comprising
DNA and RNA that may be single-stranded or, more typically,
double-stranded, or triple-stranded regions, or a mixture of
single- and double-stranded regions. In addition, "polynucleotide"
as used herein refers to triple-stranded regions comprising RNA or
DNA or both RNA and DNA. The strands in such regions may be from
the same molecule or from different molecules. The regions may
include all of one or more of the molecules, but more typically
involve only a region of some of the molecules. One of the
molecules of a triple-helical region often is an oligonucleotide.
As used herein, the term "polynucleotide(s)" also includes DNAs or
RNAs as described above that contain one or more modified bases.
Thus, DNAs or RNAs with backbones modified for stability or for
other reasons are "polynucleotide(s)" as that term is intended
herein. Moreover, DNAs or RNAs comprising unusual bases, such as
inosine, or modified bases, such as tritylated bases, to name just
two examples, are polynucleotides as the term is used herein. It
will be appreciated that a great variety of modifications have been
made to DNA and RNA that serve many useful purposes known to those
of skill in the art. The term "polynucleotide(s)" as it is employed
herein embraces such chemically, enzymatically or metabolically
modified forms of polynucleotides, as well as the chemical forms of
DNA and RNA characteristic of viruses and cells, including, for
example, simple and complex cells. "Polynucleotide(s)" also
embraces short polynucleotides often referred to as
oligonucleotide(s). Polynucleotides can also be DNA and RNA
chimeras.
[0092] As used herein, the term "polypeptide(s)" refers to any
peptide or protein comprising two or more amino acids joined to
each other by peptide bonds or modified peptide bonds.
"Polypeptide(s)" refers to both short chains, commonly referred to
as peptides, oligopeptides and oligomers and to longer chains
generally referred to as proteins. Polypeptides may contain amino
acids other than the 20 gene-encoded amino acids. "Polypeptide(s)"
include those modified either by natural processes, such as
processing and other post-translational modifications, but also by
chemical modification techniques. Such modifications are well
described in basic texts and in more detailed monographs, as well
as in a voluminous research literature, and they are well known to
those of skill in the art. It will be appreciated that the same
type of modification may be present in the same or varying degree
at several sites in a given polypeptide. Also, a given polypeptide
may contain many types of modifications. Modifications can occur
anywhere in a polypeptide, including the peptide backbone, the
amino acid side-chains, and the amino or carboxyl termini.
Modifications include, for example, acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation, selenoylation, sulfation and
transfer-RNA mediated addition of amino acids to proteins, such as
arginylation, and ubiquitination. See, for instance:
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993); Wold, F.,
Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in Posttranslational Covalent Modification of
Proteins, B. C. Johnson, Ed., Academic Press, New York (1983);
Seifter et al., Meth. Enzymol. 182:626-646 (1990); and Rattan et
al., Protein Synthesis: Posttranslational Modifications and Aging,
Ann. N.Y. Acad. Sci. 663: 48-62(1992). Polypeptides may be branched
or cyclic, with or without branching. Cyclic, branched and branched
circular polypeptides may result from post-translational natural
processes and may be made by entirely synthetic methods, as
well.
[0093] As used herein, the term "variant(s)" refers to a
polynucleotide or polypeptide that differs from a reference
polynucleotide or polypeptide, respectively, but retains one or
more of the biological activities of the initial (e.g. non-variant)
polynucleotide or polypeptide of the present invention (e.g.
STAAU_R2). A typical variant of a polynucleotide differs in
nucleotide sequence from another reference polynucleotide. Changes
in the nucleotide sequence of the variant may or may not alter the
amino acid sequence of a polypeptide encoded by the reference
polynucleotide. Nucleotide changes may result in amino acid
substitutions, additions, deletions, and truncations in the
polypeptide encoded by the reference sequence, or in the formation
of fusion proteins, as discussed below. A typical variant of a
polypeptide differs in amino acid sequence from another reference
polypeptide. Generally, differences are limited so that the
sequences of the reference polypeptide and the variant are closely
similar overall and, in many regions, identical. A variant and
reference polypeptide may differ in amino acid sequence by one or
more substitutions, additions, deletions in any combination. A
substituted or inserted amino acid residue may or may not be one
encoded by the genetic code. The present invention also includes
variants of each of the polypeptides of the invention, that is
polypeptides that vary from the referents by conservative amino
acid substitutions whereby a residue is substituted by another with
like characteristics. Typically, such substitutions are among Val,
Leu and Ile; among Ser and Thr; among the acidic residues Asp and
Glu; and among the basic residues Lys and Arg; or aromatic residues
Phe and Tyr. Particularly preferred are variants in which 1-10,
1-5,1-3, 2-3, or 1 amino acid or amino acids are substituted,
deleted, or added in any combination. A variant of a polynucleotide
or polypeptide may be a naturally occurring such as an allelic
variant, or it may be a variant that is not known to occur
naturally. Non-naturally occurring variants of polynucleotides and
polypeptides may be made by mutagenesis techniques, by direct
synthesis, and by other recombinant methods known to skilled
artisans.
[0094] As used herein, the term "fragment", when used in reference
to a polypeptide, is a polypeptide having an amino acid sequence
that is entirely the same as part but not all of the amino acid
sequence of the polypeptide according to the invention from which
it "derives". As with S. aureus STAAU_R2 polypeptides, fragments
may be "free-standing" ("consisting of"), or comprised within a
larger polypeptide of which they form a part or region, most
preferably as a single continuous region in a single larger
polypeptide.
[0095] The term "isolated", when used in reference to a nucleic
acid means that a naturally occurring sequence has been removed
from its normal cellular (e.g., chromosomal) environment or is
synthesized in a non-natural environment (e.g., artificially
synthesized). Thus, the sequence may be in a cell-free solution or
placed in a different cellular environment. The term does not imply
that the sequence is the only nucleotide chain present, but that it
is essentially free (about 90-95% pure at least) of non-nucleotide
material naturally associated with it, and thus is distinguished
from isolated chromosomes.
[0096] The term "enriched", when used in reference to a
polynucleotide means that the specific DNA or RNA sequence
constitutes a significantly higher fraction (2-5 fold) of the total
DNA or RNA present in the cells or solution of interest than in
normal or diseased cells or in cells from which the sequence was
originally taken. This could be caused by a person, by preferential
reduction in the amount of other DNA or RNA present, or by a
preferential increase in the amount of the specific DNA or RNA
sequence, or by a combination of the two. However, it should be
noted that enriched does not imply that there are no other DNA or
RNA sequences present, just that the relative amount of the
sequence of interest has been significantly increased.
[0097] As used herein, the term "significantly higher fraction"
indicates that the level of enrichment is useful to the person
making such an enrichment and indicates an increase in enrichment
relative to other nucleic acids of at least about 2-fold, or 5- to
10-fold or even more. The term also does not imply that there is no
DNA or RNA from other sources. The other source of DNA may, for
example, comprise DNA from a yeast or bacterial genome, or a
cloning vector such as pUC19. This term distinguishes from
naturally occurring events, such as viral infection, or tumor type
growths, in which the level of one mRNA may be naturally increased
relative to other species of mRNA. That is, the term is meant to
cover only those situations in which a person has intervened to
elevate the proportion of the desired nucleic acid.
[0098] As used herein, the term "purified" in reference to nucleic
acid does not require absolute purity (such as a homogeneous
preparation). Instead, it represents an indication that the
sequence is relatively more pure than in the natural environment
(compared to the natural level, this level should be at least 2-5
fold greater, e.g., in terms of mg/mL). Individual clones isolated
from a genomic or cDNA library may be purified to electrophoretic
homogeneity. The claimed DNA molecules obtained from these clones
could be obtained directly from total DNA or from total RNA. cDNA
clones are not naturally occurring, but rather are preferably
obtained via manipulation of a partially purified naturally
occurring substance (messenger RNA). The construction of a cDNA
library from mRNA involves the creation of a synthetic substance
(cDNA) and pure individual cDNA clones can be isolated from the
synthetic library by clonal selection of the cells carrying the
cDNA library. Thus, the process which includes the construction of
a cDNA library from mRNA and isolation of distinct cDNA clones
yields an approximately 10.sup.6-fold purification of the native
message over its proportion in naturally occurring cells. Thus,
purification of at least one order of magnitude, preferably two or
three orders, and more preferably four or five orders of magnitude
is expressly contemplated. A genomic library can be used in the
same way and yields the same approximate levels of
purification.
[0099] The terms "isolated", "enriched", and "purified" used with
respect to nucleic acids, above, may similarly be used to denote
the relative purity and abundance of polypeptides. These, too, may
be stored in, grown in, screened in, and selected from libraries
using biochemical techniques familiar in the art. Such polypeptides
may be natural, synthetic or chimeric and may be extracted using
any of a variety of methods, such as antibody immunoprecipitation,
other "tagging" techniques, conventional chromatography and/or
electrophoretic methods. Some of the above utilize the
corresponding nucleic acid sequence.
[0100] As used herein, the term "complement" when used in reference
to a given polynucleotide sequence refers to a sequence of
nucleotides which can form a double-stranded heteroduplex in which
every nucleotide in the sequence of nucleotides is base-paired by
hydrogen bonding to a nucleotide opposite it in the heteroduplex
with the given polynucleotide sequence. The term may refer to a DNA
or an RNA sequence that is the complement of another RNA or DNA
sequence. As used herein, the term "hybridizes" refers to the
formation of a hydrogen-bonded heteroduplex between two nucleic
acid molecules. Generally, a given nucleic acid molecule will
hybridize with its complement, or with a molecule that is
sufficiently complementary to the given molecule to permit
formation of a hydrogen-bonded heteroduplex between the two
molecules.
[0101] As used herein, the term "probe" refers to a polynucleotide
of at least 15 nucleotides (nt), 20 nt, 30 nt, 40 nt, 50 nt, 75 nt,
100 nt, 200 nt, 500 nt, 1000 nt, and even up to 5000 to 10,000 nt
in length.
[0102] "Identity" and "similarity," as used herein and as known in
the art, are relationships between two or more polypeptide
sequences or two or more polynucleotide sequences, as the case may
be, as determined by comparing the sequences.
[0103] Amino acid or nucleotide sequence "identity" and
"similarity" are determined from an optimal global alignment
between the two sequences being compared. A non-limiting example of
optimal global alignment can be carried-out using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48:443453). "Identity" means that an amino acid or nucleotide
at a particular position in a first polypeptide or polynucleotide
is identical to a corresponding amino acid or nucleotide in a
second polypeptide or polynucleotide that is in an optimal global
alignment with the first polypeptide or polynucleotide. In contrast
to identity, "similarity" encompasses amino acids that are
conservative substitutions.
[0104] The term "conservative" substitution is well-known in the
art and broadly refers to a substitution which does not
significantly change the chemico-physical properties of the
substituted amino acid. For example, a "conservative" substitution
is any substitution that has a positive score in the blosum62
substitution matrix (Hentikoff and Hentikoff, 1992, Proc. Natl.
Acad. Sci. USA 89:10915-10919). By the statement "sequence A is n %
similar to sequence B" is meant that n % of the positions of an
optimal global alignment between sequences A and B consists of
conservative substitutions. By the statement "sequence A is n %
identical to sequence B" is meant that n % of the positions of an
optimal global alignment between sequences A and B consists of
identical residues or nucleotides. Optimal global alignments in
this disclosure used the following parameters in the
Needleman-Wunsch alignment algorithm:
[0105] For polypeptides:
[0106] Substitution matrix: blosum62.
[0107] Gap scoring function: -A-B*LG, where A=11 (the gap penalty),
B=1 (the gap length penalty) and LG is the length of the gap.
[0108] For nucleotide sequences:
[0109] Substitution matrix: 10 for matches, 0 for mismatches.
[0110] Gap scoring function: -A-B*LG where A=50 (the gap penalty),
B=3 (the gap length penalty) and LG is the length of the gap.
[0111] The term `identity` and `similarity` between sequences can
be extended to their fragments. An optimal local alignment between
sequences A and B is the highest scoring alignment of fragments of
A and B. By the statement "sequence A is n % identical locally to
B" is meant that n % of the positions of an optimal local alignment
between sequences A and B consists of conservative substitutions.
By the statement "sequence A is n % similar locally to B" is meant
that n % of the position of an optimal local alignment between
sequences A and B consists of identical residues or nucleotides. An
non-limiting example of optimal local alignment can be carried-out
using the Smith-Waterman algorithm (Smith, T. F and Waterman, M. S.
1981. Identification of common molecular subsequences. J. of M.
Biol. 147:195-197).
[0112] Of course, the above-listed parameters are but one specific
example of alignment algorithm parameters. Numerous algorithms and
parameters are available and known to the person of ordinary
skill.
[0113] Typical conservative substitutions are among Met, Val, Leu
and lie; among Ser and Thr; among the residues Asp, Glu and Asn;
among the residues Gln, Lys and Arg; or aromatic residues Phe and
Tyr. In calculating the degree (most often as a percentage) of
similarity between two polypeptide sequences, one considers the
number of positions at which identity or similarity is observed
between corresponding amino acid residues in the two polypeptide
sequences in relation to the entire lengths of the two molecules
being compared.
[0114] As used herein, the term "antibody" is meant to encompass
constructions using the binding (variable) region of such an
antibody, and other antibody modifications. Thus, an antibody
useful in the invention may comprise a whole antibody, an antibody
fragment, a polyfunctional antibody aggregate, or in general a
substance comprising one or more specific binding sites from an
antibody. The antibody fragment may be a fragment such as an Fv,
Fab or F(ab').sub.2 fragment or a derivative thereof, such as a
single chain Fv fragment. The antibody or antibody fragment may be
non-recombinant, recombinant or humanized. The antibody may be of
an immunoglobulin isotype, e.g., IgG, IgM, and so forth. In
addition, an aggregate, polymer, derivative and conjugate of an
immunoglobulin or a fragment thereof can be used where appropriate.
Neutralizing antibodies are especially useful according to the
invention for diagnostics, therapeutics and methods of drug
screening and drug design.
[0115] As used herein, the term "specific for an epitope present on
a S. aureus STAAU_R2 polypeptide", when used in reference to an
antibody, means that the antibody recognizes and binds an antigenic
determinant present on a S. aureus STAAU_R2 polypeptide according
to the invention.
[0116] As used herein, the term "antigenically equivalent
derivative(s)" encompasses a polypeptide, polynucleotide, or the
equivalent of either which will be specifically recognized by
certain antibodies which, when raised to the protein, polypeptide
or polynucleotide according to the invention, interferes with the
immediate physical interaction between pathogen and mammalian
host.
[0117] As used herein, the term "essential", when used in
connection with a gene or gene product, means that the host cannot
survive without, or is significantly growth compromised, in the
absence or depletion of functional product. An "essential gene" is
thus one that encodes a product that is beneficial, or preferably
necessary, for cellular growth in vitro in a medium appropriate for
growth of a strain having a wild-type allele corresponding to the
particular gene in question. Therefore, if an essential gene is
inactivated or inhibited, that cell will grow significantly more
slowly than a wild-type strain or even not at all. Preferably,
growth of a strain in which such a gene has been inactivated will
be less than 20%, more preferably less than 10%, most preferably
less than 5% of the growth rate of the wild-type, or the rate will
be zero, in the growth medium. Preferably, in the absence of
activity provided by a product of the gene, the cell will not grow
at all or will be non-viable, at least under culture conditions
similar to normal in vivo growth conditions. For example, absence
of the biological activity of certain enzymes involved in bacterial
cell wall synthesis can result in the lysis of cells under normal
osmotic conditions, even though protoplasts can be maintained under
controlled osmotic conditions. Preferably, but not necessarily, if
such a gene is inhibited, e.g., with an antibacterial agent or a
phage product, the growth rate of the inhibited bacteria will be
less than 50%, more preferably less than 30%, still more preferably
less than 20%, and most preferably less than 10% of the growth rate
of the uninhibited bacteria. As recognized by those skilled in the
art, the degree of growth inhibition will generally depend upon the
concentration of the inhibitory agent. In the context of the
invention, essential genes are generally the preferred targets of
antimicrobial agents. Essential genes can encode "target" molecules
directly or can encode a product involved in the production,
modification, or maintenance of a target molecule.
[0118] As used herein, "target" refers to a biomolecule or complex
of biomolecules that can be acted on by an exogenous agent or
compound, thereby modulating, preferably inhibiting, growth or
viability of a bacterial cell. A target may be a nucleic acid
sequence or molecule, or a polypeptide or a region of a
polypeptide.
[0119] As used herein, the term "signal that is generated by
interaction of a S. aureus polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, or fragments thereof to a 44AHJD ORF 25,
Twort ORF 168 or G1 ORF 240 or fragment thereof" or the like refers
to the measurable indicator of polypeptide comprising the amino
acid sequence of SEQ ID NO: 2, or fragments thereof and 44AHJD ORF
25, Twort ORF 168 or G1 ORF 240 or fragment thereof interaction in
a binding assay. As used herein, the term "signal that is generated
by activation or inhibition of a S. aureus polypeptide comprising
the amino acid sequence of SEQ ID NO: 2, or fragments thereof"
refers to the measurable indicator of polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or fragments thererof,
activity in an assay of STAAU_R2 activity. For example, the signal
may include, but is not limited to (i) a signal resulting from
binding of 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240 to a STAAU_R2
polypeptide, including a fluorescence signal (fluorescence
resonance energy transfer assay; fluorescence polarization assay),
spectrophotometer absorbance measurement of a colourimetric signal
(phage display ELISA), mass change measurement (surface plasmon
resonance analysis), or a viability measurement on selective medium
(yeast two-hybrid analysis); or (ii) a reduction of a radiolabeled
signal (DNA synthesis assay).
[0120] As used herein, the term "standard", used in reference to
polypeptide activity, means the amount of activity observed or
detected (directly or indirectly) in a given assay performed in the
absence of a candidate compound. A "standard" serves as a reference
to determine the effect, positive or negative, of a candidate
compound on polypeptide activity.
[0121] As used herein, the term "increase in activity" refers to an
enhanced level of measurable activity of a polypeptide in a given
assay in the presence of a candidate compound relative to the
measurable level of activity in the absence of a candidate
compound. Activity is considered increased according to the
invention if it is at least 10% greater, 20% greater, 50% greater,
75% greater, 100% greater or more, up to 2-fold, 5-fold, 10-fold,
20-fold, 50-fold, 100-fold or more than in the absence of a
candidate compound.
[0122] As used herein, the term "decrease in activity" refers to a
reduced level of measurable activity of a polypeptide in a given
assay in the presence of a candidate compound relative to the
measurable level of activity in the absence of a candidate
compound. Activity is considered decreased according to the
invention if it is at least 10% less, preferably 15% less, 20%
less, 50% less, 75% less, or even 100% less (i.e., no activity)
than that observed in the absence of a candidate compound.
[0123] As used herein, the term "conditions that permit their
interaction", when used in reference to a S. aureus polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, or fragments
thereof, and a candidate compound means that the two entities are
placed together, whether both in solution or with one immobilized
or restricted in some way and the other in solution, wherein the
parameters (e.g., salt, detergent, protein or candidate compound
concentration, temperature, and redox potential, among others) of
the solution are such that the S. aureus polypeptide comprising the
amino acid sequence of SEQ ID NO: 2, or fragments thereof, and the
candidate compound may physically associate. Conditions that permit
protein:candidate interaction include, for example, the conditions
described herein for FRET, fluorescent polarization, Surface
Plasmon Resonance and Phage display assays.
[0124] As used herein, the term "detectable change in a measurable
parameter of STAAU_R2" refers to an alteration in a quantifiable
characteristic of a S. aureus STAAU_R2 polypeptide.
[0125] As used herein, the term "agonist" refers to an agent or
compound that enhances or increases the activity of a S. aureus
STAAU_R2 polypeptide or polynucleotide. An agonist may be directly
active on a S. aureus STAAU_R2 polypeptide or polynucleotide, or it
may be active on one or more constituents in a pathway that leads
to enhanced or increased activity of a S. aureus STAAU_R2
polypeptide or polynucleotide.
[0126] As used herein, the term "antagonist" refers to an agent or
compound that reduces or decreases the activity of a S. aureus
STAAU_R2 polypeptide or polynucleotide. An antagonist may be
directly active on a S. aureus STAAU_R2 polypeptide or
polynucleotide, or it may be active on one or more constituents in
a pathway that leads to reduced or decreased activity of a S.
aureus STAAU_R2 polypeptide or polynucleotide.
[0127] As used herein, the term "antibacterial agent" or
"antibacterial compound" refers to an agent or compound that has a
bacteriocidal or bacteriostatic effect on one or more bacterial
strains, preferably such an agent or compound is bacteriocidal or
bacteriostatic on at least S. aureus.
[0128] As used herein, the term "synthesizing" refers to a process
of chemically synthesizing a compound.
[0129] As used in the context of treating a bacterial infection a
"therapeutically effective amount", "pharmaceutically effective
amount" or "amount sufficient to provide a therapeutic effect"
indicates an amount of an antibacterial agent which has a
therapeutic effect. This generally refers to the inhibition, to
some extent, of the normal cellular functioning of bacterial cells
required for continued bacterial infection. Further, as used
herein, a therapeutically effective amount means an amount of an
antibacterial agent that produces the desired therapeutic effect as
judged for example by clinical trial results and/or animal models.
This amount can be routinely determined by one skilled in the art
and will vary depending on several factors, such as the particular
bacterial strain involved and the particular antibacterial agent
used. In the same context, an "amount sufficient to reduce
adhesion" of a bacterium to a tissue or tissue surface indicates an
amount of an antibacterial agent that is effective for
prophylactically preventing or reducing the extent of bacterial
infection of the given tissue or tissue surface.
[0130] As used in the context of treating a bacterial infection,
contacting or administering the antimicrobial agent `in combination
with existing antimicrobial agents` refer to a concurrent
contacting or administration of the active compound with
antibiotics to provide a bactericidal or growth inhibitory effects
beyond the individual bactericidal or growth inhibitory effects of
the active compound or the antibiotic. Existing antibiotic refers
for example to the group consisting of penicillins, cephalosporins,
imipenem, monobactams, aminoglycosides, tetracyclines,
sulfonamides, trimethoprim/sulfonamide, fluoroquinolones,
macrolides, vancomycin, polymyxins, chloramphenicol and
lincosamides.
[0131] As used herein, a "tissue" refers to an aggregation of cells
of one or more cell types which together perform one or more
specific functions in an organism. As used herein, a "tissue
surface" refers to that portion of a tissue that forms a boundary
between a given tissue and other tissues or the surroundings of the
tissue. A tissue surface may refer to an external surface of an
animal, for example the skin or cornea, or, alternatively, the term
may refer to a surface that is either internal, for example, the
lining of the gut, or to a surface that is exposed to the outside
surroundings of the animal only as the result of an injury or a
surgical procedure.
[0132] As used herein, the term "measuring the binding of a
candidate compound" refers to the use of an assay permitting the
quantitation of the amount of a candidate compound physically
associated with a S. aureus STAAU_R2 polypeptide, fragment or
variant thereof.
[0133] A "candidate compound" as used herein, is any compound with
a potential to modulate the expression or activity of a S. aureus
STAAU_R2 polypeptide.
[0134] As used herein, the term "directly or indirectly detectably
labeled" refers to the attachment of a moiety to a candidate
compound that renders the candidate compound either directly
detectable (e.g., an isotope or a fluorophore) or indirectly
detectable (e.g., an enzyme activity, allowing detection in the
presence of an appropriate substrate, or a specific antigen or
other marker allowing detection by addition of an antibody or other
specific indicator).
[0135] A "method of screening" refers to a method for evaluating a
relevant activity or property of a large plurality of compounds,
rather than just one or a few compounds. For example, a method of
screening can be used to conveniently test at least 100, more
preferably at least 1000, still more preferably at least 10,000,
and most preferably at least 100,000 different compounds, or even
more. In a particular embodiment, the method is amenable to
automated, cost-effective high throughput screening on libraries of
compounds for lead development.
[0136] In a related aspect or in preferred embodiments, the
invention provides a method of screening for potential
antibacterial agents by determining whether any of a plurality of
compounds, preferably a plurality of small molecules, is active on
STAAU_R2. Preferred embodiments include those described for the
above aspect, including embodiments which involve determining
whether one or more test compounds bind to or reduce the level of
activity of a bacterial target, and embodiments which utilize a
plurality of different targets as described above.
[0137] The term "compounds" preferably includes, but is not limited
to, small organic molecules, peptides, polypeptides and antibodies
that bind to a polynucleotide and/or polypeptide of the invention,
such as for example inhibitory ORF gene product or target thereof,
and thereby inhibit, extinguish or enhance its activity or
expression. Potential compounds may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or
antibody that binds the same site(s) on a binding molecule, such as
a bacteriophage gene product, thereby preventing bacteriophage gene
product from binding to STAAU_R2 polypeptides.
[0138] The term "compounds" also potentially includes small
molecules that bind to and occupy the binding site of a
polypeptide, thereby preventing binding to cellular binding
molecules, such that normal biological activity is prevented.
Examples of small molecules include but are not limited to small
organic molecules, peptides or peptide-like molecules. Preferred
potential compounds include compounds related to and variants of
inhibitory ORF encoded by a bacteriophage and of STAAU_R2 and any
homologues and/or peptidomimetics and/or fragments thereof. Other
examples of potential polypeptide antagonists include antibodies
or, in some cases, oligonucleotides or proteins which are closely
related to the ligands, substrates, receptors, enzymes, etc., as
the case may be, of the polypeptide, e.g., a fragment of the
ligands, substrates, receptors, enzymes, etc.; or small molecules
which bind to the polypeptide of the present invention but do not
elicit a response, so that the activity of the polypeptide is
prevented. Other potential compounds include antisense molecules
(see Okano, 1991 J. Neurochem. 56, 560; see also
"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression",
CRC Press, Boca Raton, Fla. (1988), for a description of these
molecules).
[0139] As used herein, the term "library" refers to a collection of
100 compounds, preferably of 1000, still more preferably 5000,
still more preferably 10,000 or more, and most preferably of 50,000
or more compounds.
[0140] As used herein, the term "small molecule" refers to
compounds having molecular mass of less than 3000 Daltons,
preferably less than 2000 or 1500, still more preferably less than
1000, and most preferably less than 600 Daltons. Preferably but not
necessarily, a small molecule is not an oligopeptide.
[0141] As used herein, the term "mimetic" refers to a compound that
can be natural, synthetic, or chimeric and is structurally and
functionally related to a reference compound. In terms of the
present invention, a "peptidomimetic," for example, is a
non-peptide compound that mimics the activity-related aspects of
the 3-dimensional structure of a peptide or polypeptide, for
example a compound that mimics the structure of a peptide or active
portion of a phage- or bacterial ORF-encoded polypeptide.
[0142] As used herein, the term "bacteriophage inhibitor protein"
refers to a protein encoded by a bacteriophage nucleic acid
sequence, which inhibits bacterial function in a host bacterium.
Thus, it is a bacteria-inhibiting phage product. The term
"bacteriophage inhibitor protein" encompasses a fragment,
derivative, or active portion of a bacteriophage inhibitor
protein.
[0143] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
STAAU_R2 or its target molecule or ligand to facilitate separation
of complexed from uncomplexed forms of one or both of the proteins
or polypeptides, as well as to accommodate automation of the assay.
Binding of a test compound to a STAAU_R2 protein or interaction of
a STAAU_R2 protein with a target molecule or ligand in the presence
and absence of a candidate compound, can be accomplished in any
vessel suitable for containing the reactants. Examples of such
vessels include microtitre plates, test tubes and micro-centrifuge
tubes. In one embodiment a fusion protein can be provided which
adds a domain that allows one or both of the proteins to be bound
to a matrix. For example, glutathione-S-transferase/STAAU_R2 fusion
proteins or glutathione-S-transferase/target fusion proteins (e.g.
glutathione-S-transferase/Twort ORF168) can be adsorbed onto
glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtitre plates, which are then combined
with the test compound or the test compound and either the
non-adsorbed target protein or STAAU_R2 protein and the mixture
incubated under conditions conducive to complex formation (e.g. at
physiological conditions for salt and pH). Following incubation the
beads or microtitre plate wells are washed to remove any unbound
components, the matrix immobilized in the case of beads, complex
determined either directly or indirectly, for example, as described
above. Alternatively, the complexes can be dissociated from the
matrix, and the level of STAAU_R2 binding or activity determined
using standard techniques.
[0144] Other techniques for immobilizing proteins on matrices (and
well-known in the art) can also be used in the screening assays of
the invention. For example, either a STAAU_R2 protein or a STAAU_R2
target molecule or ligand can be immobilized utilizing conjugation
of biotin and streptavidin. Biotinylated STAAU_R2 protein or target
molecules or ligand can be prepared from
biotin-NHS(N-hydroxy-succinimide) using techniques known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with CI-MPR
protein, target molecules or ligand but which do not interfere with
binding of the STAAU_R2 protein to its target molecule or ligand
can be derivatized to the wells of the plate, and unbound target or
STAAU_R2 protein trapped in the wells by antibody conjugation.
Methods for detecting such complexes, in addition to those
described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
STAAU_R2 protein or target molecule, as well as enzyme-linked
assays which rely on detecting an enzymatic activity associated
with the STAAU_R2 protein or target molecule and in particular with
Twort ORF168, 44AHJD ORF25, and G1 ORF 240.
[0145] As used herein, the term "active portion" refers to an
epitope, a catalytic or regulatory domain, or a fragment of a
bacteriophage inhibitor protein that is responsible for, or a
significant factor in, bacterial target inhibition. The active
portion preferably may be removed from its contiguous sequences
and, in isolation, still effect inhibition.
[0146] As used herein, the term "treating a bacterial infection"
refers to a process whereby the growth and/or metabolic activity of
a bacterium or bacterial population in a host, preferably a mammal,
more preferably a human, is inhibited or ablated.
[0147] As used herein, the term "bacterium" refers to a single
bacterial strain and includes a single cell and a plurality or
population of cells of that strain unless clearly indicated to the
contrary. In reference to bacteria or bacteriophage, the term
"strain" refers to bacteria or phage having a particular genetic
content. The genetic content includes genomic content as well as
recombinant vectors. Thus, for example, two otherwise identical
bacterial cells would represent different strains if each contained
a vector, e.g., a plasmid, with different inserts.
[0148] As used herein, the term "diagnosing" refers to the
identification of an organism or strain of an organism responsible
for a bacterial infection.
[0149] As used herein, the term "infection with Staphylococcus
aureus " refers to the presence, growth or proliferation of cells
of a S. aureus strain within, or on a surface of, an animal, such
as a mammal, preferably a human.
[0150] As used herein, the term "bacteriophage 44AHJD ORF
25-encoded polypeptide" refers to a polypeptide encoded by SEQ ID
NO: 3 or to a fragment or derivative thereof encompassing an active
portion of a bacteriophage 44AHJD ORF 25-encoded polypeptide of
sequence disclosed in SEQ ID NO: 4.
[0151] As used herein, the term "bacteriophage Twort ORF168-encoded
polypeptide" refers to a polypeptide encoded by SEQ ID NO: 5 or to
a fragment or derivative thereof encompassing an active portion of
a bacteriophage Twort ORF168-encoded polypeptide of sequence
disclosed in SEQ ID NO: 6.
[0152] As used herein, the term "bacteriophage G1 ORF 240-encoded
polypeptide" refers to a polypeptide encoded by SEQ ID NO: 9 or to
a fragment or derivative thereof encompassing an active portion of
a bacteriophage G1 ORF 240-encoded polypeptide of sequence
disclosed in SEQ ID NO: 10.
[0153] As used herein, the term "polypeptide complex" refers to a
combination of two or more polypeptides in a physical association
with each other. It is preferred that such a physical association
be required for some aspect of the activity of one or more of the
polypeptides in such a polypeptide complex.
[0154] As used herein, the term "physical association" refers to an
interaction between two moieties involving contact between the two
moieties.
[0155] As used herein, the term "bodily material(s)" means any
material derived from an individual or from an organism infecting,
infesting or inhabiting an individual, including but not limited
to, cells, tissues and waste, such as, bone, blood, serum,
cerebrospinal fluid, semen, saliva, muscle, cartilage, organ
tissue, skin, urine, stool or autopsy materials.
[0156] As used herein, the term "disease(s)" means any disease
caused by or related to infection by a bacterium, including, for
example, otitis media, conjunctivitis, pneumonia, bacteremia,
meningitis, sinusitis, pleural empyema and endocarditis, and most
particularly meningitis, such as for example infection of
cerebrospinal fluid.
[0157] As used herein, the term "fusion protein(s)" refers to a
protein encoded by a gene comprising amino acid coding sequences
from two or more separate proteins fused in frame such that the
protein comprises fused amino acid sequences from the separate
proteins.
[0158] As used herein, the term "host cell(s)" is a cell which has
been transformed or transfected, or is capable of transformation or
transfection by an exogenous polynucleotide sequence.
[0159] As used herein, the term "immunologically equivalent
derivative(s)" encompasses a polypeptide, polynucleotide, or the
equivalent of either which when used in a suitable formulation to
raise antibodies in a vertebrate, results in antibodies that act to
interfere with the immediate physical interaction between pathogen
and mammalian host.
[0160] As used herein, the term "immunospecific" means that
characteristic of an antibody whereby it possesses substantially
greater affinity for the polypeptides of the invention or the
polynucleotides of the invention than its affinity for other
related polypeptides or polynucleotides respectively, particularly
those polypeptides and polynucleotides in the prior art.
[0161] As used herein, the term "individual(s)" means a
multicellular eukaryote, including, but not limited to a metazoan,
a mammal, an ovid, a bovid, a simian, a primate, and a human.
[0162] As used herein, the term "Organism(s)" means a (i)
prokaryote, including but not limited to, a member of the genus
Streptococcus, Staphylococcus, Bordetella, Corynebacterium,
Mycobacterium, Neisseria, Haemophilus, Actinomycetes,
Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,
Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,
Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium,
Brucella, Bacillus, Clostridium, Treponema, Escherichia,
Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia,
Leptospira, Spirillum, Campylobacter, Shigella, Legionella,
Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and
Mycoplasma, and further including, but not limited to, a member of
the species or group, Group A Streptococcus, Group B Streptococcus,
Group C Streptococcus, Group D Streptococcus, Group G
Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,
Streptococcus agalactiae, Streptococcus faecalis, Streptococcus
faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria
meningitidis, Staphylococcus aureus, Staphylococcus epidermidis,
Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium
tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans,
Mycobacterium leprae, Actinomyctes israeli, Listeria monocytogenes,
Bordetella pertusis, Bordatella parapertusis, Bordetella
bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus
influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae,
Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter
freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis,
Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens,
Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas
aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus
anthracis, Bacillus cereus, Clostridium perfringens, Clostridium
tetani, Clostridium botulinum, Treponema pallidum, Rickettsia
rickettsii and Chlamydia trachomitis, (ii) an archaeon, including
but not limited to Archaebacter, and (iii) a unicellular or
filamenous eukaryote, including but not limited to, a protozoan, a
fungus, a member of the genus Saccharomyces, Kluveromyces, or
Candida, and a member of the species Saccharomyces ceriviseae,
Kluveromyces lactis, or Candida albicans.
[0163] As used herein, the term "recombinant expression system(s)"
refers to a system in which vectors comprising sequences encoding
polypeptides of the invention or portions thereof, or
polynucleotides of the invention are introduced or transformed into
a host cell or host cell lysate for the production of the
polynucleotides and polypeptides of the invention.
[0164] As used herein, the term "artificially synthesized" when
used in reference to a peptide, polypeptide or polynucleotide means
that the amino acid or nucleotide subunits were chemically joined
in vitro without the use of cells or polymerizing enzymes. The
chemistry of polynucleotide and peptide synthesis is well known in
the art.
[0165] In addition to the standard single and triple letter
representations for amino acids, the term "X" or "Xaa" may also be
used in describing certain polypeptides of the invention. "X" and
"Xaa" mean that any of the twenty naturally occurring amino acids
may appear at such a designated position in the polypeptide
sequence.
[0166] As used herein, the term "specifically binding" in the
context of the interaction of two polypeptides means that the two
polypeptides physically interact via discrete regions or domains on
the polypeptides, wherein the interaction is dependent upon the
amino acid sequences of the interacting domains. Generally, the
equilibrium binding concentration of a polypeptide that
specifically binds another is in the range of about 1 mM or lower,
more preferably 1 uM or lower, preferably 100 nM or lower, 10 nM or
lower, 1 nM or lower, 100 pM or lower, and even 10 pM or lower.
[0167] As used herein, the term "decrease in the binding" refers to
a drop in the signal that is generated by the physical association
between two polypeptides under one set of conditions relative to
the signal under another set of reference conditions. The signal is
decreased if it is at least 10% lower than the level under
reference conditions, and preferably 20%, 40%, 50%, 75%, 90%, 95%
or even as much as 100% lower (i.e., no detectable
interaction).
[0168] As used herein, the term "detectable marker", when used in
the context of a yeast two-hybrid assay, refers to a polypeptide
that confers a trait upon a cell expressing that polypeptide that
signals the presence or amount of that polypeptide expressed.
Detectable markers are encoded on plasmids that may exist
episomally or may be integrated into the genome of a host cell.
Detectable markers include, but are not limited to, polypeptides
encoding enzymes allowing colorimetric or fluorescent detection
(e.g., E. coli LacZ, which catalyzes the conversion of the
substrate analog X-gal to generate a blue color), polypeptides
encoding enzymes conferring antibiotic resistance, and polypeptides
encoding enzymes conferring the ability of a yeast strain to grow
on medium lacking a given component (i.e., critical for the relief
of auxotrophy).
[0169] As used herein, the term "results in the expression of a
detectable marker" means that the interaction of factors necessary
to permit the expression of a detectable marker (e.g., two-hybrid
transactivation domain and DNA binding domain fusion proteins)
causes the transactivation and translation of detectable levels of
a detectable marker. A "detectable level" is that level of
expression that can be differentiated from background expression
occurring in the substantial absence of one or more factors or
conditions necessary for marker expression. Detectable levels will
vary depending upon the nature of the detectable marker, but will
generally consist of levels at least about 10% or more greater than
the background level of a given marker.
[0170] As used herein, the term "decrease in the expression" refers
to a drop in the expression of a detectable marker under one set of
conditions relative to the expression under another set of
reference conditions. The expression of a detectable marker is
decreased if it is at least 10% lower than the level under
reference conditions, and preferably 20%, 40%, 50%, 75%, 90%, 95%
or even as much as 100% lower (i.e., not expressed).
[0171] Identification of the S. aureus STAAU_R2 Sequence
[0172] The methodology used to identify the STAAU_R2 polypeptide is
described in detail in U.S. Provisional Patent Application No.
60/110,992, filed Dec. 3,1998, and PCT International Application
WO1999/IB99/02040, filed Dec. 3, 1999.
[0173] A S. aureus polypeptide that specifically bound the
bacterial growth inhibitory 44AHJD ORF 25, Twort ORF 168 or G1 ORF
240 proteins was isolated. Briefly, the three inhibitory ORF
proteins were used separately as a ligand in an affinity
chromatography binding step with S. aureus protein extract.
[0174] The selected S. aureus interacting polypeptide, herein
referred as STAAU_R2, was purified and further analyzed by tryptic
digestion and mass spectrometry using MALDI-ToF technology [Qin,
J., et al. (1997) Anal. Chem. 69, 3995-4001]. Computational
analysis of the mass spectrum obtained identified the corresponding
ORF as the S. aureus DNA polymerase III, beta subunit. The
interaction between Twort ORF 168 and the STAAU_R2, was confirmed
in a yeast two-hybrid assay. The interaction between 44AHJD ORF 25
and the candidate target protein was confirmed by surface plasmon
resonance.
[0175] Function of the DNA Polymerase III, Beta (.beta.)
Subunit
[0176] DNA polymerase III holoenzyme is an essential component of
bacterial DNA replication machinery. The holoenzyme contains
several different polypeptide chains. Type III polymerases are
exemplified by the replicase of the Gram-negative bacterium
Escherichia coli, in which there are three separate components: a
sliding clamp protein, a clamp loader complex and the DNA
polymerase itself [Kelman et al. 1995, Annu. Rev. Biochem. 64:
171-200]. The clamp loader is a multiprotein complex which uses ATP
to assemble the sliding clamp around DNA. The DNA polymerase then
binds to the sliding clamp which tethers the polymerase to the DNA.
The DNA polymerase III beta (.beta.) subunit is a homodimer and
forms the ring shaped sliding clamp associated with DNA.
[0177] Bacillus subtilis and Streptococcus pyogenes are the best
characterized Gram-positive bacteria with respect to DNA
replication [Barnes et al. 1995, Methods in Enzy. 262: 35-42; Bruck
and O'Donnell 2000, J. Biol. Chem. 275: 28971-28983]. Many genes
involved in Bacillus subtilis DNA replication have been identified
through the isolation of ts mutants. Studies in B. subtilis have
identified a polymerase that appears to be involved in chromosome
replication and is termed Pol III. The polC gene encodes Pol III, a
large polypeptide likely corresponding to the alpha and epsilon
subunits of the E. coli enzyme. In in vitro reconstituted assays,
five Streptococcus pyogenes proteins were shown to coordinate their
actions to achieve rapid and processive DNA synthesis: the PolC DNA
polymerase, .tau., .delta., .delta.' and .beta. [Bruck and
O'Donnell 2000, J. Biol. Chem. 275: 28971-28983].
[0178] S. aureus has a gene encoding a protein with 30% homology to
the .beta. of the E. coli enzyme. The S. aureus gene corresponding
to the E. coli .beta. subunit is dnaN. S. aureus dnaN has been
described in two PCT Applications (WO 9937661; and WO 0109164).
[0179] The E. coli .beta. subunit has been shown to interact with a
variety of proteins including DNA polymerase III .alpha. and
.delta. subunits, DNA polymerase I, DNA polymerase II, DNA
polymerase V, DNA ligase and MutS [Jeruzalmi et al. 2001, Cell,
106: 417-428; Lopez de Saro and O'Donnell 2001, PNAS, 98:
8376-8380]. Surprisingly, despite the demonstration of
protein-protein interactions of the .beta. subunit with a variety
of proteins and despite evidence that these interactions within the
replisome are critical to obtaining efficient chromosome
replication in vitro [Turner et al. 1999, The EMBO J. 18: 771-783;
Jeruzalmi et al. 2001, Cell, 106: 417-428; Bruck and O'Donnell
2000, J. Biol. Chem. 275: 28971-28983], there are currently no
available drugs directed against the sliding clamp.
[0180] The demonstration that bacteriophage have adapted to
inhibiting a host bacterium by acting on a particular cellular
component or target provides a strong indication that that
component is an appropriate target for developing and using
antibacterial agents, e.g. in therapeutic treatments. The present
invention provides additional guidance over mere identification of
bacterial essential genes, as the present invention also provides
an indication of accessibility of the target to an inhibitor, and
an indication that the target is sufficiently stable over time
(e.g., not subject to high rates of mutation) as phage acting on
that target were able to develop and persist. Thus, the present
invention identifies STAAU_R2 as an appropriate target for
development of antibacterial agents.
[0181] Identification of the Surface of Interaction on STAAU_R2
[0182] This invention relates, in part, to a specific interaction
between a growth-inhibitory protein encoded by the S. aureus
bacteriophage genome and an essential S. aureus protein. In one
embodiment, this interaction forms the basis for drug screening
assays. More specifically, the invention relates to the interacting
regions of the protein encoded by the S. aureus STAAU_R2 and the S.
aureus bacteriophage 44AHJD ORF 25, Twort ORF 168, G1 ORF 240
proteins, forming the basis for screening assays. The invention
provides a method for the identification of 44AHJD ORF 25, Twort
ORF 168 (e.g. with SEQ ID NO: 8) or G1 ORF 240 and, more
preferably, STAAU_R2 polypeptide fragments which are involved in
the interaction between STAAU_R2 and 44AHJD ORF 25, Twort ORF 168,
G1 ORF 240.
[0183] Several approaches and techniques known to those skilled in
the art can be used to identify and to characterize interacting
fragments of STAAU_R2, 44AHJD ORF 25, Twort ORF 168, G1 ORF 240.
These fragments may include, for example, truncation polypeptides
having a portion of an amino acid sequence of any of the two
proteins, or variants thereof, such as a continuous series of
residues that includes an amino- and/or carboxyl-terminal amino
acid sequence.
[0184] Fragments of STAAU_R2,44AHJD ORF 25, Twort ORF 168, G1 ORF
240_can be cloned by genetic recombinant technology and tested for
interaction using a yeast two-hybrid assay as exemplified
below.
[0185] Partial proteolysis of proteins in solution is one method to
delineate the domain boundaries in multi-domain proteins. By
subjecting proteins to limited digestion, the most accessible
cleavage sites are preferentially hydrolyzed. These cleavage sites
preferentially reside in less structured regions which include
loops and highly mobile areas typical of the joining amino acids
between highly structures domains. Purified STAAU_R2, 44AHJD ORF
25, Twort ORF 168, G1 ORF 240_proteins can be subjected to partial
proteolysis. The proteolysis can be performed with low
concentrations of proteases (trypsin, chymotrypsin, endoproteinase
Glu-C, and Asp-N) with STAAU_R2, 44AHJD ORF 25, Twort ORF 168, G1
ORF 240 in solution, resulting in the generation of defined
proteolytic products as observed by SDS-PAGE. An acceptable
concentration and reaction time is defined by the near complete
conversion of the full-length protein to stable proteolytic
products. The proteolytic products are then subjected to affinity
chromatography containing the appropriated partner of interaction
(44AHJD ORF 25, Twort ORF 168; G1 ORF 240_or STAAU_R2 purified
proteins) to determine a protein sub-region able to interact.
Interacting domains are identified by mass spectrometry to
determine both the intact fragment mass and the completely digested
with trypsin (by in-gel digestion) to better determine the amino
acid residues contained within the partial proteolytic fragment.
Using both sets of data, the amino acid sequence of the partial
proteolytic fragment can be precisely determined.
[0186] Another approach is based on peptide screening using
different portions of 44AHJD ORF 25, Twort ORF 168, G1 ORF 240 or
STAAU_R2 to identify minimal peptides from each polypeptide that
are able to disrupt the interaction between the two proteins. It is
assumed that fragments able to prevent interaction between STAAU_R2
and 44AHJD ORF 25, Twort ORF 168, or G1 ORF 240 correspond to
domains of interaction located on either of the two interacting
proteins. The different peptide fragments can be screened as
competitors of interaction in protein: protein binding assays such
as the ones described below. Fine mapping of interaction site(s)
within a protein can be performed by an extensive screen of small
overlapping fragments or peptides spanning the entire amino acid
sequence of the protein.
[0187] Suitable STAAU_R2, Twort ORF 168, 44AHJD ORF 25, G1 ORF
240--derived amino acid fragments representative of the complete
sequence of both proteins can be chemical synthesis. For instance,
in the multipin approach, peptides are simultaneously synthesis by
the assembly of small quantities of peptides (ca. 50 nmol) on
plastic pins derivatized with an ester linker based on glycolate
and 4-(hydroxymethyl) benzoate (Maeji 1991 Pept Res, 4:142-6).
[0188] S. aureus STAAU_R2 Polypeptides
[0189] In one aspect of the invention there are provided
polypeptides of S. aureus referred to herein as "STAAU_R2" and
"STAAU_R2 polypeptides" as well as biologically, diagnostically,
prophylactically, clinically or therapeutically useful variants
thereof, and compositions comprising the same.
[0190] Among the particularly preferred embodiments of the
invention are variants of S. aureus STAAU_R2 polypeptides encoded
by naturally occurring alleles of the STAAU_R2 gene. The present
invention provides for an isolated polypeptide which comprises or
consists of: (a) an amino acid sequence which has at least 40%
identity, preferably at least 50% identity, preferably at least 80%
identity, more preferably at least 90%, yet more preferably at
least 95%, most preferably at least 97-99%, or exact identity, over
the entire length of SEQ ID NO: 2; or b) an amino acid sequence
that has at least 60% similarity, at least 70% similarity, at least
80% similarity, at least 90% similarity, at least 95% similarity,
at least 97-99% similarity or even 100% similarity over the entire
length of SEQ ID NO: 2.
[0191] The polypeptides of the invention include a polypeptide of
FIG. 1 (SEQ ID NO: 2) (in particular the mature polypeptide) as
well as polypeptides and fragments, particularly those which have a
biological activity of STAAU_R2, and also those which have at least
40% identity over 50 or more amino acids to a polypeptide of SEQ ID
NO: 2 or the relevant portion, preferably at least 60%, 70%, or 80%
identity over 50 or more amino acids to a polypeptide of SEQ ID NO:
2, more preferably at least 90% identity over 50 or more amino
acids to a polypeptide of SEQ ID NO: 2 and still more preferably at
least 95% identity over 50 or more amino acids to a polypeptide of
SEQ ID NO: 2 and yet still more preferably at least 99% identity
over 50 or more amino acids to a polypeptide of SEQ ID NO: 2.
[0192] The polypeptides of the invention also include a polypeptide
or protein fragment that has at least 60%, 70%, 80% or 90%
similarity, 95% similarity or even 97-99% similarity over 50 or
more amino acids to a polypeptide of SEQ ID NO: 2.
[0193] It is most preferred that a polypeptide of the invention is
derived from S. aureus, however, it may be obtained from other
organisms of the same taxonomic genus. A polypeptide of the
invention may also be obtained, for example, from organisms of the
same taxonomic family or order.
[0194] Fragments of STAAU_R2 also are included in the invention.
These fragments may include, for example, truncation polypeptides
having a portion of an amino acid sequence of FIG. 1 (SEQ ID NO:
2), or variants thereof, such as a continuous series of residues
that includes an amino- and/or carboxyl-terminal amino acid
sequence. Degradation forms of the polypeptides of the invention
produced by or in a host cell, particularly S. aureus, are also
preferred. Further preferred are fragments characterized by
structural or functional attributes such as fragments that comprise
alpha-helix and alpha-helix-forming regions, beta-sheet and
beta-sheet-forming regions, turn and turn-forming regions, coil and
coil-forming regions, hydrophilic regions, hydrophobic regions,
alpha amphipathic regions, beta amphipathic regions, flexible
regions, surface-forming regions, substrate binding region, and
high antigenic index regions. Fragments of STAAU_R2 may be
expressed as fusion proteins with other proteins or protein
fragments.
[0195] Preferred fragments also include an isolated polypeptide
comprising an amino acid sequence having at least 10, 20, 30, 40,
50, or 100 or more contiguous amino acids from the amino acid
sequence of SEQ ID NO: 2.
[0196] Also preferred are biologically "active" fragments which are
those fragments that mediate activities of S. aureus STAAU_R2,
including those with a similar activity or an improved activity, or
with a decreased undesirable activity. Also included are those
fragments that are antigenic or immunogenic in an animal,
especially in a human. Particularly preferred are fragments
comprising domains that confer a function essential for viability
of S. aureus.
[0197] Fragments of the polypeptides of the invention may be
employed for producing the corresponding full-length polypeptide by
peptide synthesis; therefore, these variants may be employed as
intermediates for producing the full-length polypeptides of the
invention.
[0198] S. aureus Polynucleotides
[0199] It is an object of the invention to provide polynucleotides
that encode STAAU_R2 polypeptides, particularly polynucleotides
that encode the polypeptide herein designated S. aureus
STAAU_R2.
[0200] In one aspect of the invention, a polynucleotide is provided
that comprises a region encoding a S. aureus STAAU_R2 polypeptide,
the polynucleotide comprising a sequence set out in SEQ ID NO: 1.
Such a polynucleotide encodes a full length STAAU_R2 gene, or a
variant thereof. It is contemplated that this full-length gene is
essential to the growth and/or survival of an organism which
possesses it, such as S. aureus.
[0201] As a further aspect of the invention there are provided
isolated nucleic acid molecules encoding and/or expressing a
fragment of a full-length STAAU_R2 polypeptide, particularly a S.
aureus STAAU_R2 polypeptide or a variant thereof. Further
embodiments of the invention include biologically, diagnostically,
prophylactically, clinically or therapeutically useful
polynucleotides, polypeptides, variants thereof, and compositions
comprising same.
[0202] A polynucleotide of the invention is obtained using S.
aureus cells as starting material, the nucleotide sequence
information disclosed in SEQ ID NO: 1, and standard cloning and
screening methods, such as those for cloning and sequencing
chromosomal DNA fragments from bacteria. For example, to obtain a
polynucleotide sequence of the invention, such as the
polynucleotide sequence disclosed as in SEQ ID NO: 1, a library of
clones of chromosomal DNA of S. aureus in E. coli or another
suitable host is probed with a radiolabeled oligonucleotide,
preferably a 17-mer or longer, derived from a partial sequence.
Clones carrying DNA identical to that of the probe can be
distinguished using stringent hybridization conditions. As herein
used, the terms "stringent conditions" and "stringent hybridization
conditions" mean hybridization occurring only if there is at least
95% and preferably at least 97% identity between the sequences. A
specific example of stringent hybridization conditions is of an
overnight incubation of a hybridization support (e.g., a nylon or
nitrocellulose membrane) at 42.degree. C. in a solution comprising:
1.times.10.sup.6 cpm/ml labeled probe, 50% formamide, 5.times.SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20
micrograms/ml of denatured, sheared salmon sperm DNA, followed by
washing the hybridization support in 0.1.times.SSC at 65.degree. C.
Hybridization and wash conditions are well known to those skilled
in the art and are exemplified in Sambrook, et al., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor,
N.Y., (1989), particularly Chapter 11 therein. Solution
hybridization may also be used with the polynucleotide sequences
provided by the invention. By sequencing the individual clones thus
identified by hybridization, it is possible to confirm the identity
of the clone.
[0203] Alternatively, an amplification process can be utilized to
isolate the poylnucleotide. In this approach, the sequence
disclosed as SEQ ID NO: 1 is targeted by two oligonucleotides, one
identical to a sequence on the coding DNA strand at or upstream of
the ATG initiation codon and the other which anneals to the
opposite strand at or downstream of the stop codon. Priming from
these oligonucleotides in a polymerase chain reaction yields a
full-length gene coding sequence. Such suitable techniques are
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd Edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1989).
[0204] In a further aspect, the present invention provides for an
isolated polynucleotide comprising or consisting of: (a) a
polynucleotide sequence which has at least 60% identity, preferably
at least 70% identity, more preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95%,
most preferably at least 97-99% or exact identity, to that of SEQ
ID NO: 1 over the entire length of SEQ ID NO: 1; (b) a
polynucleotide sequence encoding a polypeptide which has at least
50% identity, preferably at least 60% identity, more preferably at
least 70% identity, more preferably at least 80% identity, more
preferably at least 90%, yet more preferably at least 95%, most
preferably at least 97-99% or exact identity to SEQ ID NO: 2 over
the entire length of SEQ ID NO: 2; or the complement of a sequence
of (a) or (b) above.
[0205] The invention provides a polynucleotide sequence identical
over its entire length to the coding sequence of SEQ ID NO: 1. Also
provided by the invention is a coding sequence for a mature
polypeptide or a fragment thereof by itself as well as a coding
sequence for a mature polypeptide or a fragment in reading frame
with another coding sequence, such as a sequence encoding a leader
or secretory sequence, a pre-, or pro-, or prepro-protein sequence.
The polynucleotide of the invention may also contain at least one
non-coding sequence, including for example, but not limited to at
least one non-coding 5' and 3' sequence, such as the transcribed
but non-translated sequences, termination signals (such as
rho-dependent and rho-independent termination signals), ribosome
binding sites, Kozak sequences, sequences that stabilize or
destabilize mRNAs, introns, and polyadenylation signals. The
polynucleotide sequence may also comprise additional coding
sequence encoding additional amino acids. For example, a marker
sequence that facilitates purification of the fused polypeptide can
be encoded. In certain embodiments of the invention, the marker
sequence is a hexa-histidine peptide, as provided in the pQE vector
(Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.
Sci. 86: 821-824 (1989), or an HA peptide tag [Wilson et al., Cell
37: 767 (1984)], both of which may-be useful in purifying
polypeptide sequences fused to them. Polynucleotides of the
invention also include, but are not limited to, polynucleotides
comprising a structural gene and its naturally associated sequences
that control gene expression.
[0206] While it is most preferred that a polynucleotide of the
invention be derived from S. aureus, it may also be obtained from
other organisms of the same taxonomic genus. A polynucleotide of
the invention may also be obtained, for example, from organisms of
the same taxonomic family or order.
[0207] Further preferred embodiments are polynucleotides encoding
S. aureus STAAU_R2 variants that have the amino acid sequence of S.
aureus STAAU_R2 polypeptide of SEQ ID NO: 2 in which several, a
few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are
substituted, modified, deleted and/or added, in any combination.
Especially preferred among these polynucleotides are those encoding
silent nucleotide alterations that do not alter the coding sequence
or activities of S. aureus STAAU_R2 polypeptides they encode.
[0208] Preferred embodiments are polynucleotides encoding
polypeptides that retain substantially the same biological function
or activity as the mature polypeptide encoded by a DNA of SEQ ID
NO: 1.
[0209] In accordance with certain preferred embodiments of this
invention there are provided polynucleotides that hybridize,
particularly under stringent conditions, to S. aureus STAAU_R2
polynucleotide sequences, such as those polynucleotides in FIG.
1.
[0210] The polynucleotides of the invention are useful as
hybridization probes for RNA, cDNA and genomic DNA to isolate
full-length cDNAs and genomic clones encoding genes that have a
high degree of sequence identity to the STAAU_R2 gene. Such probes
generally will comprise at least 15 to about 100 residues or base
pairs, although such probes will preferably have about 20 to 50
nucleotide residues or base pairs. Particularly preferred probes
are about 20 to about 30 nucleotide residues or base pairs in
length.
[0211] A coding region of a related STAAU_R2 gene from a bacterial
species other than S. aureus may be isolated by screening a library
using a DNA sequence provided in SEQ ID NO: 1 to synthesize an
oligonucleotide probe. A labeled oligonucleotide having a sequence
complementary to that of a gene of the invention is then used to
screen a library of cDNA, genomic DNA or mRNA to determine to which
member(s) of the library the probe hybridizes.
[0212] There are several methods available and well known to those
skilled in the art to obtain full-length DNAs, or extend short
DNAs, for example those based on the method of Rapid Amplification
of cDNA Ends (RACE) [see, for example, Frohman, et al., PNAS USA
85: 8998-9002, 1988]. Recent modifications of the technique,
exemplified by the MARATHON.TM. technology (Clontech Laboratories
Inc.) for example, have significantly simplified the search for
longer cDNAs. In the MARATHON.TM. technology, cDNAs are prepared
from mRNA extracted from a chosen cell and an `adaptor` sequence is
ligated onto each end. Nucleic acid amplification by PCR is then
carried out to amplify the "missing" 5' end of the DNA using a
combination of gene specific and adaptor specific oligonucleotide
primers. The PCR reaction is then repeated using "nested" primers,
that is, primers designed to anneal within the amplified product
(typically an adaptor-specific primer that anneals further 3' in
the adaptor sequence and a gene-specific primer that anneals
further 5' in the selected gene sequence). The products of this
reaction can then be analyzed by DNA sequencing and a full-length
DNA constructed either by joining the product directly to the
existing DNA to give a complete sequence, or by carrying out a
separate full-length PCR using the new sequence information for the
design of the 5' primer.
[0213] The polynucleotides and polypeptides of the invention may be
employed, for example, as research reagents and materials for
discovery of treatments of and diagnostics for diseases,
particularly human diseases, as further discussed herein relating
to polynucleotide assays.
[0214] The polynucleotides of the invention that are
oligonucleotides derived from a sequence of SEQ ID NO:1 are useful
for the design of PCR primers in reactions to determine whether or
not the polynucleotides identified herein in whole or in part are
transcribed in bacteria in infected tissue. That is, the
polynucleotides of the invention are useful for diagnosis of
infection with a bacterial strain carrying those sequences. It is
recognized that such sequences also have utility in diagnosis of
the stage of infection and type of infection the pathogen has
attained.
[0215] The invention also provides polynucleotides that encode a
polypeptide that is the mature protein plus additional amino or
carboxyl-terminal amino acids, or amino acids interior to the
mature polypeptide. Such sequences may play a role in processing of
a protein from precursor to a mature form, may allow protein
transport, may lengthen or shorten protein half-life or may
facilitate manipulation of a protein for assay or production, among
other things. As generally is the case in vivo, the additional
amino acids may be processed away from the mature protein by
cellular enzymes.
[0216] A precursor protein, having a mature form of the polypeptide
fused to one or more prosequences may be an inactive form of the
polypeptide. When prosequences are removed such inactive precursors
generally are activated. Some or all of the prosequences may be
removed before activation. Generally, such precursors are called
proproteins.
[0217] A polynucleotide of the invention thus may encode a mature
protein, a mature protein plus a leader sequence (which may be
referred to as a preprotein), a precursor of a mature protein
having one or more prosequences that are not the leader sequences
of a preprotein, or a preproprotein, which is a precursor to a
proprotein, having a leader sequence and one or more prosequences,
which generally are removed during processing steps that produce
active and mature forms of the polypeptide.
[0218] In addition to the standard A, G, C, T/U representations for
nucleotides, the term "N" may also be used in describing certain
polynucleotides of the invention. "N" means that any of the four
DNA or RNA nucleotides may appear at such a designated position in
the DNA or RNA sequence, except it is preferred that N is not a
nucleotide that when taken in combination with adjacent nucleotide
positions, read in the correct reading frame, would have the effect
of generating a premature termination codon in such reading
frame.
[0219] For each and every polynucleotide of the invention there is
also provided a polynucleotide complementary to it.
[0220] Vectors, Host Cells, and Expression Systems
[0221] The invention also relates to vectors that comprise a
polynucleotide or polynucleotides of the invention, host cells that
are genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the invention
[0222] Recombinant STAAU_R2 polypeptides of the present invention
may be prepared by processes well known to those skilled in the art
from genetically engineered host cells comprising expression
systems. Accordingly, in a further aspect, the present invention
relates to expression systems that comprise a STAAU_R2
polynucleotide or polynucleotides of the present invention, to host
cells which are genetically engineered with such expression
systems, and to the production of polypeptides of the invention by
recombinant techniques.
[0223] For recombinant production of STAAU_R2 polypeptides of the
invention, host cells can be genetically engineered to incorporate
expression systems or portions thereof or polynucleotides of the
invention. Representative examples of appropriate hosts include
bacterial cells (Gram positive and Gram negative), fungal cells,
insect cells, animal cells and plant cells. Polynucleotides are
introduced to bacteria by standard chemical treatment protocols,
such as the induction of competence to take up DNA by treatment
with calcium chloride (Sambrook et al., supra). Introduction of
polynucleotides into fungal (e.g., yeast) host cells is effected,
if desired, by standard chemical methods, such as lithium
acetate--mediated transformation.
[0224] A great variety of expression systems are useful to produce
STAAU_R2 polypeptides of the invention. Such vectors include among
others, chromosomal-, episomal- and virus-derived vectors. For
example, vectors derived from bacterial plasmids, from
bacteriophages, from transposons, from yeast episomes, from
insertion elements, from yeast chromosomal elements, from viruses,
and from vectors derived from combinations thereof, are useful in
the invention.
[0225] STAAU_R2 polypeptides of the invention are recovered and
purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid or urea
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, and lectin chromatography. Well known techniques
for refolding may be employed to regenerate an active conformation
when the STAAU_R2 polypeptide is denatured during isolation and/or
purification.
[0226] Diagnostic, Prognostic, Serotyping, and Mutation Assays
[0227] This invention is also related to the use of STAAU_R2
polynucleotides and polypeptides of the invention for use as
diagnostic reagents. Detection of S. aureus STAAU_R2
polynucleotides and/or polypeptides in a eukaryote, particularly a
mammal, and especially a human, will provide a diagnostic method
for diagnosis of disease, staging of disease or response of an
infectious organism to drugs. Eukaryotes, particularly mammals, and
especially humans, particularly those infected or suspected to be
infected with an organism comprising the S. aureus STAAU_R2 gene or
protein, may be detected at the nucleic acid or amino acid level by
a variety of well known techniques as well as by methods provided
herein.
[0228] Polypeptides and polynucleotides for prognosis, diagnosis or
other analysis may be obtained from a putatively infected and/or
infected individual's bodily materials. Polynucleotides from any of
these sources, particularly DNA or RNA, may be used directly for
detection or may be amplified enzymatically by using PCR or any
other amplification technique prior to analysis. RNA, particularly
mRNA, cDNA and genomic DNA may also be used in the same ways. Using
amplification, characterization of the species and strain of
infectious or resident organism present in an individual, may be
made by an analysis of the genotype of a selected polynucleotide of
the organism. Deletions and insertions can be detected by a change
in size of the amplified product in comparison to a genotype of a
reference sequence selected from a related organism, preferably a
different species of the same genus or a different strain of the
same species.
[0229] Point mutations can be identified by hybridizing amplified
DNA to labeled STAAU_R2 polynucleotide sequences. Perfectly or
significantly matched sequences can be distinguished from
imperfectly or more significantly mismatched duplexes by DNase or
RNase digestion, for DNA or RNA respectively, or by detecting
differences in melting temperatures or renaturation kinetics.
Polynucleotide sequence differences may also be detected by
alterations in the electrophoretic mobility of polynucleotide
fragments in gels as compared to a reference sequence. This may be
carried out with or without denaturing agents. Polynucleotide
differences may also be detected by direct DNA or RNA sequencing.
See, for example, Myers et al, (1985) Science 230, 1242. Sequence
changes at specific locations also may be revealed by nuclease
protection assays, such as RNase, V1 and S1 protection assay or a
chemical cleavage method. See, for example, Cotton et al., (1985)
Proc. Natl. Acad. Sci., USA 85, 4397-4401.
[0230] In another embodiment, an array of oligonucleotide probes
comprising STAAU_R2 nucleotide sequence or fragments thereof can be
constructed to conduct efficient screening of, for example, genetic
mutations, serotype, taxonomic classification or identification.
Array technology methods are well known and have general
applicability and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and
genetic variability (see, for example, Chee et al., (1996) Science
274, 610).
[0231] Thus in another aspect, the present invention relates to a
diagnostic kit which comprises: (a) a polynucleotide of the present
invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a
fragment thereof; (b) a nucleotide sequence complementary to that
of (a); (c) a polypeptide of the present invention, preferably the
polypeptide of SEQ ID NO: 2 or a fragment thereof; or (d) an
antibody to a polypeptide of the present invention, preferably to
the polypeptide of SEQ ID NO: 2 or fragment thereof.
[0232] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component. Such a kit will be of
use in diagnosing a disease or susceptibility to a disease, among
others.
[0233] This invention also relates to the use of STAAU_R2
polynucleotides of the present invention as diagnostic reagents.
Detection of a mutated form of a polynucleotide of the invention,
preferably, SEQ ID NO: 1, which is associated with a disease or
pathogenicity will provide a diagnostic tool that can add to, or
define, a diagnosis of a disease, a prognosis of a course of
disease, a determination of a stage of disease, or a susceptibility
to a disease, which results from under-expression, over-expression
or altered expression of the polynucleotide. Organisms,
particularly infectious organisms, carrying mutations in such
polynucleotide may be detected at the polynucleotide level by a
variety of techniques, such as those described elsewhere
herein.
[0234] The STAAU_R2 nucleotide sequences of the present invention
are also valuable for organism chromosome identification. The
sequence is specifically targeted to, and can hybridize with, a
particular location on an organism's chromosome, particularly to a
S. aureus chromosome. The mapping of relevant sequences to
chromosomes according to the present invention may be an important
step in correlating those sequences with pathogenic potential
and/or an ecological niche of an organism and/or drug resistance of
an organism, as well as the essentiality of the gene to the
organism. Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data may be found
on-line in a sequence database. The relationship between genes and
diseases that have been mapped to the same chromosomal region are
then identified through known genetic methods, for example, through
linkage analysis (coinheritance of physically adjacent genes) or
mating studies, such as by conjugation.
[0235] The differences in a polynucleotide and/or polypeptide
sequence between organisms possessing a first phenotype and
organisms possessing a different, second different phenotype can
also be determined. If a mutation is observed in some or all
organisms possessing the first phenotype but not in any organisms
possessing the second phenotype, then the mutation is likely to be
the causative agent of the first phenotype.
[0236] Polypeptides and polynucleotides for prognosis, diagnosis or
other analysis may be obtained from a putatively infected and/or
infected individual's bodily materials. Particularly DNA or
polynucleotides, from any of these sources may be used directly for
detection or may be amplified enzymatically using PCR or other
amplification technique with oligonucleotide amplification primers
derived from the polynucleotide sequence of S. aureus STAAU_R2.
RNA, particularly mRNA, or RNA reverse transcribed to cDNA, is also
useful for diagnostics. Following amplification of a S. aureus
STAAU_R2-related polynucleotide from a sample, characterization of
the species and strain of infecting or resident organism is made by
an analysis of the amplified polynucleotide relative to one or more
reference polynucleotides or sequences relative to a standard from
a related organism (i.e. a known strain of S. aureus).
[0237] The invention further provides a process for diagnosing
bacterial infections such as those caused by S. aureus, the process
comprising determining from a sample derived from an individual,
such as a bodily material, an increased level of expression of a
polynucleotide having a sequence disclosed in SEQ ID NO: 1 relative
to a sample taken from a non-diseased individual. Increased or
decreased expression of a STAAU_R2 polynucleotide can be measured
using any one of the methods well known in the art for the
quantitation of polynucleotides, such as, for example, PCR, RT-PCR,
RNase protection, Northern blotting and other hybridization
methods, and spectrometry.
[0238] In addition, a diagnostic assay in accordance with the
invention for detecting over-expression of STAAU_R2 polypeptide
compared to normal control tissue samples may be used to detect the
presence of an infection, for example. Assay techniques that can be
used to determine levels of a S. aureus DnaN polypeptide, in a
sample derived from a host, such as a bodily material, are
well-known to those of skill in the art. Such assay methods include
radioimmunoassays, competitive-binding assays, Western Blot
analysis, antibody sandwich assays, antibody detection and ELISA
assays.
[0239] Gridding and Polynucleotide Subtraction of S. aureus Genomic
Sequences
[0240] The STAAU_R2 polynucleotides of the invention may be used as
components of polynucleotide arrays, preferably high density arrays
or grids. These high density arrays are particularly useful for
diagnostic and prognostic purposes. For example, a set of spots
each comprising a different gene, and further comprising a
polynucleotide or polynucleotides of the invention, may be used for
probing, such as hybridization or nucleic acid amplification, using
a probe obtained or derived from a bodily sample, to determine the
presence a particular polynucleotide sequence or related sequence
in an individual.
[0241] Antibodies Specific for S. aureus Peptides or
Polypeptides
[0242] The STAAU_R2 polypeptides and polynucleotides of the
invention or variants thereof, or cells expressing them are useful
as immunogens to produce antibodies immunospecific for such
polypeptides or polynucleotides, respectively.
[0243] In certain preferred embodiments of the invention there are
provided antibodies against S. aureus STAAU_R2 polypeptides or
polynucleotides encoding them. Antibodies against
STAAU_R2-polypeptide or STMU R2-polynucleotide are useful for
treatment of infections, particularly bacterial infections.
[0244] Antibodies generated against the polypeptides or
polynucleotides of the invention are obtained by administering the
polypeptides and/or polynucleotides of the invention or
epitope-bearing fragments of either or both, analogues of either or
both, or cells expressing either or both, to an animal, preferably
a nonhuman, using routine protocols. For preparation of monoclonal
antibodies, any technique known in the art that provides antibodies
produced by continuous cell line cultures is useful. Examples
include various techniques, such as those in Kohler, G. and
Milstein, C., Nature 256: 495497 (1975); Kozbor et al., Immunology
Today 4: 72 (1983); and Cole et al., pg. 96-96 in Monoclonal
Anbitodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
[0245] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,968) can be adapted to produce single chain
antibodies to polypeptides or polynucleotides of this invention.
Also, transgenic mice, or other mammals, are useful to express
humanized antibodies immunospecific to the polypeptides or
polynucleotides of the invention.
[0246] When antibodies are administered therapeutically, the
antibody or variant thereof is preferably modified to make it less
immunogenic in the individual. For example, if the individual is
human the antibody is most preferably "humanized," where the
complementarity determining region or regions of the
hybridoma-derived antibody has been transplanted into a human
monoclonal antibody, for example as described in Jones et al.
(1986), Nature 321, 522-525 or Tempest et al., (1991) Biotechnology
9, 266-273.
[0247] Alternatively, phage display technology is useful to select
antibody genes with binding activities towards a STAAU_R2
polypeptide of the invention. In one possible scheme, antibody
fragments specific for S. aureus STAAU_R2 are selected from an
immune library of antibody genes expressed as fusions with coat
protein of filamentous phage. Alternatively, naive libraries are
screened by phage display techniques to identify genes encoding
antibodies specified for STAAU_R2 or from naive libraries
[McCafferty, et al., (1990), Nature 348, 552-554; Marks, et al.,
(1992) Biotechnology 10, 969-783; a recent reference is de Haard et
al. (1999) J Biol Chem 274: 18218-18230]. The ability to recover,
for various targets, antibodies with subnanomolar affinities
obviates the need for immunization. The affinity of these
antibodies can also be improved by, for example, chain shuffling
[Clackson et al., (1991 j Nature 352: 628].
[0248] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptides or polynucleotides
of the invention, for example to purify the polypeptides or
polynucleotides by immunoaffinity chromatography.
[0249] A variant polypeptide or polynucleotide of the invention,
such as an antigenically or immunologically equivalent derivative
or a fusion protein of the polypeptide is also useful as an antigen
to immunize a mouse or other animal such as a rat or chicken. A
fused protein provides stability to the polypeptide acting as a
carrier, or acts as an adjuvant or both. Alternatively, the antigen
is associated, for example by conjugation, with an immunogenic
carrier protein, such as bovine serum albumin, keyhole limpet
haemocyanin or tetanus toxoid. Alternatively, when antibodies are
to be administered therapeutically, alternatively a multiple
antigenic polypeptide comprising multiple copies of the
polypeptide, or an antigenically or immunologically equivalent
polypeptide thereof may be sufficiently antigenic to improve
immunogenicity so as to obviate the use of a carrier.
[0250] In accordance with an aspect of the invention, there is
provided the use of a STAAU_R2 polynucleotide of the invention for
therapeutic or prophylactic purposes, in particular genetic
immunization. The use of a STAAU_R2 polynucleotide of the invention
in genetic immunization preferably employs a suitable delivery
method such as direct injection of plasmid DNA into muscles [Wolff
et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene
Ther. (1983) 4: 419], delivery of DNA complexed with specific
protein carriers [Wu et al., J Biol. Chem. (1989).264: 16985],
coprecipitation of DNA with calcium phosphate [Benvenisty &
Reshef, PNAS USA, (1986) 83: 9551], encapsulation of DNA in various
forms of liposomes [Kaneda et al., Science (1989) 243: 375],
particle bombardment [Tang et al., Nature (1992) 356:152,
Eisenbraun et al., DNA Cell Biol (1993) 12: 791] or in vivo
infection using cloned retroviral vectors [Seeger et al., PNAS USA
(1984) 81: 5849].
[0251] Antagonists and Agonists: Assays and Molecules
[0252] The invention is based in part on the discovery that
STAAU_R2 is a target for the bacteriophage 44AHJD ORF 25, Twort
ORF168, G1 ORF 240 inhibitory factors. Applicants have recognized
the utility of the interaction in the development of antibacterial
agents. Specifically, the inventors have recognized that 1)
STAAU_R2 is a critical target for bacterial inhibition; 2) 44AHJD
ORF 25, Twort ORF 168, G1 ORF 240 or derivatives or functional
mimetics thereof are useful for inhibiting bacterial growth; and 3)
the interaction between STAAU_R2 of S. aureus and 44AHJD ORF 25,
Twort ORF 168, G1 ORF 240 may be used as a target for the screening
and rational design of drugs or antibacterial agents. In addition
to methods of directly inhibiting STAAU_R2 activity, methods of
inhibiting STAAU_R2 expression are also attractive for
antibacterial activity.
[0253] In several embodiments of the invention, there are provided
methods for identifying compounds which bind to or otherwise
interact with and inhibit or activate an activity or expression of
a polypeptide and/or polynucleotide of the invention comprising:
contacting a polypeptide and/or polynucleotide of the invention
with a compound to be screened under conditions to permit binding
to or other interaction between the compound and the polypeptide
and/or polynucleotide to assess the binding to or other interaction
with the compound, such binding or interaction preferably being
associated with a second component capable of providing a
detectable signal in response to the binding or interaction of the
polypeptide and/or polynucleotide with the compound; and
determining whether the compound binds to or otherwise interacts
with and activates or inhibits an activity or expression of the
polypeptide and/or polynucleotide by detecting the presence or
absence of a signal generated from the binding or interaction of
the compound with the polypeptide and/or polynucleotide.
[0254] Potential antagonists include, among others, small organic
molecules, peptides, polypeptides and antibodies that bind to a
polynucleotide and/or polypeptide of the invention and thereby
inhibit or extinguish its activity or expression. Potential
antagonists also may be small organic molecules, a peptide, a
polypeptide such as a closely related protein or antibody that
binds the same sites on a binding molecule, such as a binding
molecule, without inducing STAAU_R2-induced activities, thereby
preventing the action or expression of S. aureus STAAU_R2
polypeptides and/or polynucleotides by excluding S. aureus STAAU_R2
polypeptides and/or polynucleotides from binding.
[0255] Potential antagonists also include a small molecule that
binds to and occupies the binding site of the polypeptide thereby
preventing binding to cellular binding molecules, such that normal
biological activity is prevented. Cellular binding molecules
include but are not limited to proteins involved in DNA
replication. Examples of cellular binding molecules include DNA
polymerase III (x and 6 subunits, DNA polymerase 1, DNA polymerase
11, DNA polymerase V, DNA ligase and MutS polypeptides.
[0256] Examples of small molecules include but are not limited to
small organic molecules, peptides or peptide-like molecules. Other
potential antagonists include antisense molecules [see Okano,
(1991) J. Neurochem. 56, 560; see also Oligodeoxynucleotides As
Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton,
Fla. (1988), for a description of these molecules]. Preferred
potential antagonists include compounds related to and variants of
44AHJD ORF 25, Twort ORF 168, G1 ORF 240 and of STAAU_R2. Other
examples of potential polypeptide antagonists include antibodies
or, in some cases, oligonucleotides or proteins which are closely
related to the ligands, substrates, receptors, enzymes, etc., as
the case may be, of the polypeptide, e.g., a fragment of the
ligands, substrates, receptors, enzymes, etc.; or small molecules
which bind to the polypeptide of the present invention but do not
elicit a response, so that the activity of the polypeptide is
prevented.
[0257] Compounds may be identified from a variety of sources, for
example, cells, cell-free preparations, chemical libraries, and
natural product mixtures. These substrates and ligands may be
natural substrates and ligands or may be structural or functional
mimetics. See, e.g., Coligan et al., Current Protocols in
Immunology 1(2): Chapter 5 (1991). Peptide modulators can also be
selected by screening large random libraries of all possible
peptides of a certain length.
[0258] Compounds derived from the polypeptide sequence of 44AHJD
ORF25, Twort ORF 168 or G1 ORF 240 could represent fragments
representing small overlapping peptides spanning the entire amino
acid sequence of these ORFs. Fragments of 44AHJD ORF25, Twort ORF
168 or G1 ORF 240 can be produced as described above.
[0259] Certain of the polypeptides of the invention are
biomimetics, functional mimetics of the natural S. aureus STAAU_R2
polypeptide. These functional mimetics are useful for, among other
things, antagonizing the activity of S. aureus STAAU_R2 polypeptide
or as an antigen or immunogen in a manner described above.
Functional mimetics of the polypeptides of the invention include
but are not limited to truncated polypeptides. For example,
preferred functional mimetics include a polypeptide comprising the
polypeptide sequence set forth in SEQ ID NO: 2 lacking 20, 30, 40,
50, 100, 200, 300, 325 amino- or carboxy-terminal amino acid
residues, including fusion proteins comprising one or more of these
truncated sequences. Polynucleotides encoding each of these
functional mimetics may be used as expression cassettes to express
each mimetic polypeptide. It is preferred that these cassettes
comprise-5' and 3' restriction sites to allow for a convenient
means to ligate the cassettes together when desired. It is further
preferred that these cassettes comprise gene expression signals
known in the art or described elsewhere herein.
[0260] Screening Assays According to the Invention
[0261] It is desirable to devise screening methods to identify
compounds which stimulate or which inhibit the function of the
STAAU_R2 polypeptide or polynucleotide of the invention.
Accordingly, the present invention provides for a method of
screening compounds to identify those that modulate the function of
a polypeptide or polynucleotide of the invention. In general,
antagonists may be employed for therapeutic and prophylactic
purposes. It is contemplated that an agonist of STAAU_R2 may be
useful, for example, to enhance the growth rate of bacteria in a
sample being cultured for diagnostic or other purposes.
[0262] It has been determined that STAAU_R2 is a target for
bacteriophage 44AHJD ORF 25, Twort ORF168 and G1 ORF 240 products,
which acts as inhibitory factors. Applicants have recognized the
utility of the interaction in the development of antibacterial
agents. Polypeptide and/or polynucleotide targets such as STAAU_R2
are critical targets for bacterial inhibition. S. aureus
bacteriophage 44AHJD ORF 25, Twort ORF168, G1 ORF 240_or
derivatives or functional mimetics thereof are useful for
inhibiting bacterial growth and the interaction, binding,
inhibition and/or activation which occurs between polypeptides,
such as for example STAAU_R2 of S. aureus and 44AHJD ORF 25, Twort
ORF168, G1 ORF 240 may be used for the screening and rational
design of drugs or antibacterial agents. In addition to methods for
directly inhibiting a target such as STAAU_R2 activity, methods of
inhibiting a target such as STAAU_R2 expression are also attractive
for antibacterial activity.
[0263] In preferred embodiments, the method involves the
interaction of an inhibitory ORF product or fragment thereof with
the corresponding bacterial target or fragment thereof that
maintains the interaction with the ORF product or fragment.
Interference with the interaction between the components can be
monitored, and such interference is indicative of compounds that
can inhibit, activate, or enhance the activity of the target
molecule.
[0264] a. Binding Assays
[0265] There are a number of methods of examining binding of a
candidate compound to a protein target such as STAAU_R2 and a
polypeptide comprising amino acid sequence of SEQ ID NO: 2, or
fragments thereof. Screening methods that measure the binding of a
candidate compound to the STAAU_R2 polypeptide or polynucleotide,
or to cells or supports bearing the polypeptide or a fusion protein
comprising the polypeptide, by means of a label directly or
indirectly associated with the candidate compound, are useful in
the invention.
[0266] The screening method may involve competition for binding of
a labeled competitor such as 44AHJD ORF 25, Twort ORF 168, G1 ORF
240 or a fragment that is competent to bind STAAU_R2.
[0267] Non-limiting examples of screening assays [Reviewed in
Sittampalam et al. 1997 Curr Opin Chem Biol. 3:384-91] in
accordance with the present invention include the following.
[0268] i.) Fluorescence Resonance Energy Transfer (FRET)
[0269] A method of measuring inhibition of binding of two proteins
using fluorescence resonance energy transfer [FRET; de Angelis,
1999, Physiological Genomics]. FRET is a quantum mechanical
phenomenon that occurs between a fluorescence donor (D) and a
fluorescence acceptor (A) in close proximity (usually <100 A of
separation.) if the emission spectrum of D overlaps with the
excitation spectrum of A. Variants of the green fluorescent protein
(GFP) from the jellyfish Aequorea Victoria are fused to a
polypeptide or protein and serve as D-A pairs in a FRET scheme to
measure protein-protein interaction. Cyan (CFP: D) and yellow (YFP:
A) fluorescence proteins are linked with STAAU_R2 polypeptide, or a
fragment of STAAU_R2 and 44AHJD ORF 25, Twort ORF 168, G1 ORF 240
protein respectively. Under optimal proximity, interaction between
STAAU_R2, or a fragment of STAAU_R2, and 44AHJD ORF 25, Twort ORF
168, G1 ORF 240 causes a decrease in intensity of CFP fluorescence
concomitant with an increase in YFP fluorescence.
[0270] The addition of a candidate modulator to the mixture of
appropriately labeled STAAU_R2 and 44AHJD ORF 25, Twort ORF 168, G1
ORF 240 protein, will result in an inhibition of energy transfer
evidenced by, for example, a decease in YFP fluorescence at a given
concentration of 44AHJD ORF 25, Twort ORF 168, G1 ORF 240 relative
to a sample without the candidate inhibitor.
[0271] ii.) Fluorescence Polarization
[0272] Fluorescence polarization measurement is another useful
method to quantitate protein-protein binding. The fluorescence
polarization value for a fluorescently-tagged molecule depends on
the rotational correlation time or tumbling rate. Protein
complexes, such as those formed by S. aureus STAAU_R2 polypeptide,
or a fragment of STAAU_R2 associating with a fluorescently labeled
polypeptide (e.g., 44AHJD ORF 25, Twort ORF 168, G1 ORF 240 or a
binding fragment thereof), have higher polarization values than
does the fluorescently labeled polypeptide. Inclusion of a
candidate inhibitor of the STAAU_R2 interaction results in a
decrease in fluorescence polarization relative to a mixture without
the candidate inhibitor if the candidate inhibitor disrupts or
inhibits the interaction of STAAU_R2 with its polypeptide binding
partner. It is preferred that this method be used to characterize
small molecules that disrupt the formation of polypeptide or
protein complexes.
[0273] iii.) Surface Plasmon Resonance
[0274] Another powerful assay to screen for inhibitors of a
protein: protein interaction is surface plasmon resonance. Surface
plasmon resonance is a quantitative method that measures binding
between two (or more) molecules by the change in mass near a sensor
surface caused by the binding of one protein or other biomolecule
from the aqueous phase (analyte) to a second protein or biomolecule
immobilized on the sensor (ligand). This change in mass is measured
as resonance units versus time after injection or removal of the
second protein or biomolecule (analyte) and is measured using a
Biacore Biosensor (Biacore AB) or similar device. STAAU_R2, or a
polypeptide comprising fragment of STAAU_R2, could be immobilized
as a ligand on a sensor chip (for example, research grade CM5 chip;
Biacore AB) using a covalent linkage method (e.g. amine coupling in
10 mM sodium acetate [pH 4.5]). A blank surface is prepared by
activating and inactivating a sensor chip without protein
immobilization. Alternatively, a ligand surface can be prepared by
noncovalent capture of ligand on the surface of the sensor chip by
means of a peptide affinity tag, an antibody, or biotinylation. The
binding of 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240 to STAAU_R2,
or a fragment of STAAU_R2, is measured by injecting purified 44AHJD
ORF 25, Twort ORF 168 or G1 ORF 240 over the ligand chip surface.
Measurements are performed at any desired temperature between
4.degree. C. and 37.degree. C. Conditions used for the assay (i.e.,
those permitting binding) are as follows: 25 mM HEPES-KOH (pH 7.6),
150 mM sodium chloride, 15% glycerol, 1 mM dithiothreitol, and
0.001% Tween 20 with a flow rate of 10 ul/min. Preincubation of the
sensor chip with candidate inhibitors will predictably decrease the
interaction between 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240 and
STAAU_R2. A decrease in 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240
binding, detected as a reduced response on sensorgrams and measured
in resonance units, is indicative of competitive binding by the
candidate compound.
[0275] iv.) Scintillation Proximity Assay
[0276] A scintillation proximity assay (SPA) may be used to
characterize the interaction between a S. aureus STAAU_R2
polypeptide, or a fragment of STAAU_R2 polypeptide, for example
comprising the amino acid sequence of SEQ ID NO: 2, or a part
thereof, and another polypeptide. The SPA relies in a solid-phase
substrate, such as beads or the plastic of a microtitre plate, into
which a scintillant has been incorporated. For the assay, the
target protein, for example a S. aureus STAAU_R2 polypeptide, is
coupled to the beads or to the surface of the plate, either
covalently through activated surface chemistries or non-covalently
through a peptide affinity tag, an antibody, or biotinylation.
Addition of a radiolabeled binding polypeptide, for example
[.sup.32P]-radiolabeled 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240,
results in close proximity of the radioactive source molecule to
the scintillant. As a consequence, the radioactive decay excites
the scintillant contained within the bead or within the plastic of
the plate and detectable light is emitted. Compounds that prevent
the association between immobilized S. aureus STAAU_R2 polypeptide
and radiolabeled 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240 will
diminish the scintillation signal. The SPA thus represents an
example of an ideal technology with which to screen for inhibitors
of the STAAU_R244AHJD ORF 25, STAAU_R2-Twort ORF 168 or STAAU_R2-G1
ORF 240 interactions because it is readily adapted to
high-throughput, automated format and because of its sensitivity
for detection of protein-protein interactions with K.sub.D values
in the micromolar to nanomolar ranges.
[0277] v.) Bio Sensor Assay
[0278] ICS biosensors have been described by AMBRI (Australian
Membrane Biotechnology Research Institute;
http//www.ambri.com.au/). In this technology, the self-association
of macromolecules such as STAAU_R2, or a fragment of STAAU_R2, and
bacteriophage 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240, is
coupled to the closing of gramacidin-facilitated ion channels in
suspended membrane bilayers and hence to a measurable change in the
admittance (similar to impedence) of the biosensor. This approach
is linear over six order of magnitude of admittance change and is
ideally suited for large scale, high through-put screening of small
molecule combinatorial libraries.
[0279] vi.) Phage Display
[0280] Phage display is a powerful assay to measure protein:protein
interaction. In this scheme, proteins or peptides are expressed as
fusions with coat proteins or tail proteins of filamentous
bacteriophage. A comprehensive monograph on this subject is Phage
Display of Peptides and Proteins. A Laboratory Manual edited by Kay
et al. (1996) Academic Press. For phages in the Ff family that
include M13 and fd, gene III protein and gene VIII protein are the
most commonly-used partners for fusion with foreign protein or
peptides. Phagemids are vectors containing origins of replication
both for plasmids and for bacteriophage. Phagemids encoding fusions
to the gene III or gene VIII can be rescued from their bacterial
hosts with helper phage, resulting in the display of the foreign
sequences on the coat or at the tip of the recombinant phage.
[0281] In the simplest assay, purified recombinant STAAU_R2
protein, or a fragment of STAAU_R2, could be immobilized in the
wells of a microtitre plate and incubated with phages displaying
44AHJD ORF 25, Twort ORF 168 or G1 ORF 240 in fusion with the gene
III protein. Washing steps are performed to remove unbound phages
and bound phages are detected with monoclonal antibodies directed
against phage coat protein (gene VIII protein). An enzyme-linked
secondary antibody allows quantitative detection of bound fusion
protein by fluorescence, chemiluminescence, or colourimetric
conversion. Screening for inhibitors is performed by the incubation
of the compound with the immobilized target before the addition of
phages. The presence of an inhibitor will specifically reduce the
signal in a dose-dependent manner relative to controls without
inhibitor.
[0282] It is important to note that in assays of protein-protein
interaction, it is possible that a modulator of the interaction
need not necessarily interact directly with the domain(s) of the
proteins that physically interact. It is also possible that a
modulator will interact at a location removed from the site of
protein-protein interaction and cause, for example, a
conformational change in the STAAU_R2 polypeptide. Modulators
(inhibitors or agonists) that act in this manner can be termed
allosteric effectors and are of interest since the change they
induce may modify the activity of the STAAU_R2 polypeptide.
[0283] b. Assays of STAAU_R2 Functional Activity
[0284] Non-limiting examples of assays to assess the functional
enzymatic activity of STAAU_R2, or fragments thereof, variant or
homolog thereof, include the measurement of stimulation of in vitro
DNA replication. There are number of methods of measuring the DNA
synthesis stimulation of a polypeptide comprising the amino acid
sequence of STAAU_R2.
[0285] For example, an assay for STAAU_R2 activity could involved
the measurement of radiolabeled nucleotide incorporated into
cellular DNA. Samples (0.5 ml) are withdrawn from cultures at
appropriate time intervals and mixed with 4.5 .mu.l of labeling
solution (0.2 .mu.Ci/ml of .sup.3H-thymidine (73 Ci/mmol, NEN Life
Science Products, Inc.) and 70 pmol of unlabeled thymidine). After
15 minutes of reaction, incorporation is stopped by adding 5 .mu.l
of 0.2% NaN.sub.3 and 5 .mu.l of 30 .mu.g/ml unlabeled thymidine.
Samples are precipitated with 10% (w/v) trichloroacetic acid and
filtered through glass fiber filters (GF-C, Whatman). The results
are expressed as .sup.3H-thymidine counts incorporated, normalized
to the OD of the culture.
[0286] DNA synthesis could be measured by using a soluble cell-free
in vitro system based on the use of a variety of different
synthetic DNA substrates. The replication assay could involved
crude or partially purified cellular proteins extracts. The
replication assays could be reconstituted with partially pure or
pure form of native proteins or recombinantly produced
proteins.
[0287] In one cell-free in vitro assay, an extract prepared from S.
aureus is supplied to a plasmid substrate, for example a primed
circular M13ssDNA substrate, in a reaction including exogenous
radiolabeled deoxynucleotide triphosphates (dATP, dTTP, dGTP and
dCTP), MgCl.sub.2 and ATP. The reaction is stopped and the products
precipitated with trichloroacetic acid, and then filtered.
Scintillation counting of the dried filter gives the level of de
novo replication.
[0288] Another example to assay for STAAU_R2 activity is to measure
the level of radiolabeled nucleotide incorporated into DNA in a
reconstituted in vitro assay using primed circular ssDNA substrate
[Bruck and O'Donnell 2000, J. Biol. Chem. 275: 28971-28983]. The
replication reactions typically, contained Tris-HCl [pH 7.5],
MgCl.sub.2, BSA, DTT, ATP, dCTP, dGTP, and dATP,
[.alpha.-.sup.32P]dTTP, EDTA, glycerol, circular primed M13ssDNA,
S. aureus SSB, PolC, .tau., .delta. and .delta.' and increasing
amount of STAAU_R2 polypeptide. Reactions were incubated at
37.degree. C. for 5 min and quenched upon addition of SDS and EDTA.
Half of the quenched reaction is analyzed for total DNA synthesis
using a DE81 filter paper. The other half is analyzed by
electrophoresis on agarose gel. Gels are dried, and radioactive
products were visualized following exposure to a phosphorImager
screen for the evaluation of the rate of DNA synthesis and
processivity.
[0289] Testing for inhibitors, for example 44AHJD ORF 25 and Twort
ORF168, is performed by the incubation of the compound with the
reaction mixtures. The presence of an inhibitor will specifically
reduce the signal in a dose-dependent manner relative to controls
without inhibitor. Compounds selected for their ability to inhibit
interactions between STAAU_R2-44AHJD ORF 25 or STAAU_R2-Twort ORF
can be further tested in functional activity assays.
[0290] Alternatively, a rapid fluorometric assay that measures the
activity of replication enzymes could be developed to measure
STAAU_R2 activity. The fluorometric assay is based on the
preferential binding of a fluorescent dye to double stranded DNA,
for example, de novo synthesized DNA, vs. single stranded DNA and
it has been described (Seville et al., 1996. Biotechniques
21:664-672). A reconstituted in vitro assay similar to that
described using primed circular ssDNA substrate [Bruck and
O'Donnell 2000, J. Biol. Chem. 275:28971-28983] could be developed.
The replication reactions would contain Tris-HCl [pH 7.5],
MgCl.sub.2, BSA, DTT, ATP, dNTPs, EDTA, glycerol, circular primed
M13ssDNA, S. aureus SSB, PolC, .tau., .delta. and .delta.' and
increasing amount of STAAU_R2 polypeptide. Reactions are incubated
at 37.degree. C. for variable times then quenched. The quenched
reaction is analyzed for total DNA synthesis by adding
PicoGreen.TM. dye (Molecular Probes, Eugene, Oreg.), incubating 5
min at room temperature, and reading the intensity of fluorescence
of PicoGreen (.lambda..sub.EX, 485 nm; .lambda..sub.EM, 525 nm).
The sensitivity of the dye and the homogeneous nature of the
PicoGreen assay should allow rapid screening in a non-radiometric
assay format.
[0291] Testing for inhibitors, for example of 44AHJD ORF 25, Twort
ORF168 or G1 ORF 240 (of fragments or variants thereof), is
performed by the incubation of the compound with the reaction
mixtures. The presence of an inhibitor will specifically reduce the
fluorescence signal in a dose-dependent manner relative to controls
without inhibitor. Compounds selected for their ability to inhibit
interactions between STAAU_R2-44AHJD ORF 25, STAAU_R2-Twort ORF 168
or STAAU_R2-G1 ORF 240 can be further tested in functional activity
assays.
[0292] c. Bacterial Growth Inhibition
[0293] Compounds selected for their ability to inhibit interactions
between STAAU_R2 and 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240 or
to inhibit the STAAU_R2 activity can be further tested in
functional assays of bacterial growth. Cultures of S. aureus are
grown in the presence of varying concentrations of a candidate
compound added directly to the medium or using a vehicle which is
appropriate for the delivery of the compound into the cell. For
compounds that correspond to polypeptides, the nucleotide sequence
encoding said polypeptides can be cloned into a S. aureus
expression vector containing an inducible promotor. The expression
of the polypeptide could be induced following transfection of
cells. For example, the polypeptide may include, but is not limited
to the different 44AHJD ORF 25, Twort ORF 168 or G1 ORF 240-derived
fragments (e.g. with SEQ ID NO: 8).
[0294] Following the induction of expression or the addition of
compound, the cultures are then incubated for an additional 4 h at
37.degree. C. During that period of time, the effect of inhibitors
on bacterial cell growth may be monitored at 40 min intervals, by
measuring, for example, the OD.sub.565 and the number of colony
forming units (CFU) in the cultures. The number of CFU is evaluated
as follows: cultures are serially diluted and aliquots from the
different cultures are plated out on agar plates. Following
incubation overnight at 37.degree. C., the number of colonies is
counted. Non-treated cultures of S. aureus are included as negative
control.
[0295] In another aspect, the present invention relates to a
screening kit for identifying agonists, antagonists, ligands,
receptors, substrates, enzymes, etc. for a polypeptide and/or
polynucleotide of the present invention; or compounds which
decrease or enhance the production of such polypeptides and/or
polynucleotides, which comprises: (a) a polypeptide and/or a
polynucleotide of the present invention; (b) a recombinant cell
expressing a polypeptide and/or polynucleotide of the present
invention; (c) a cell membrane associated with a polypeptide and/or
polynucleotide of the present invention; or (d) an antibody to a
polypeptide and/or polynucleotide of the present invention; which
polypeptide is preferably that of SEQ ID NO: 2, and for which the
polynucleotide is preferably that of SEQ ID NO: 1.
[0296] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0297] It will be readily appreciated by the skilled artisan that a
polypeptide and/or polynucleotide of the present invention may also
be used in a method for the structure-based design of an agonist,
antagonist or inhibitor of the polypeptide and/or polynucleotide,
by: (a) determining in the first instance the three-dimensional
structure of the polypeptide and/or polynucleotide, or complexes
thereof; (b) deducing the three-dimensional structure for the
likely reactive site(s), binding site(s) or motif(s) of an agonist,
antagonist or inhibitor; (c) synthesizing candidate compounds that
are predicted to bind to or react with the deduced binding site(s),
reactive site(s), and/or motif(s); and (d) testing whether the
candidate compounds are indeed agonists, antagonists or inhibitors.
It will be further appreciated that this will normally be an
iterative process, and this iterative process may be performed
using automated and computer-controlled steps.
[0298] Each of the polynucleotide sequences provided herein may be
used in the discovery and development of antibacterial compounds.
The encoded protein, upon expression, can be used as a target for
the screening of antibacterial drugs. Additionally, the
polynucleotide sequences encoding the amino terminal regions of the
encoded protein or Shine-Dalgarno or other sequence that facilitate
translation of the respective mRNA can be used to construct
antisense sequences to control the expression of the coding
sequence of interest.
[0299] The invention also provides the use of the polypeptide,
polynucleotide, agonist or antagonist of the invention to interfere
with the initial physical interaction between a pathogen or
pathogens and a eukaryotic, preferably mammalian, host that is
responsible for sequelae of infection. In particular, the molecules
of the invention may be used: in the prevention of adhesion of
bacteria, in particular Gram positive and/or Gram negative
bacteria, to eukaryotic, preferably mammalian, extracellular matrix
proteins on in-dwelling devices or to extracellular matrix proteins
in wounds; to block bacterial adhesion between eukaryotic,
preferably mammalian, extracellular matrix proteins and bacterial
STAAU_R2 proteins that mediate tissue damage and/or; to block the
normal progression of pathogenesis in infections initiated other
than by the implantation of in-dwelling devices or by other
surgical techniques.
[0300] In accordance with yet another aspect of the invention,
there are provided STAAU_R2 antagonists, preferably bacteriostatic
or bacteriocidal antagonists.
[0301] The antagonists of the invention may be employed, for
instance, to prevent, inhibit and/or treat diseases.
[0302] Compositions, Kits and Administration
[0303] In a further aspect of the invention there are provided
compositions comprising a STAAU_R2 polynucleotide and/or a S.
aureus STAAU_R2 polypeptide for administration to a cell or to a
multicellular organism.
[0304] The present invention provides for pharmaceutical
compositions comprising a therapeutically effective amount of a
polypeptide and/or polynucleotide, such as the soluble form of a
polypeptide and/or polynucleotide of the present invention,
antagonist peptide or small molecule compound, in combination with
a pharmaceutically acceptable carrier or excipient. Such carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The
pharmaceutical compositions may be administered in any effective,
convenient manner including, for instance, administration by
topical, oral, anal, vaginal, intravenous, intraperitoneal,
intramuscular, subcutaneous, intranasal or intradermal routes among
others.
[0305] In therapy or as a prophylactic, the active agent may be
administered to an individual as an injectable composition, for
example as a sterile aqueous dispersion, preferably isotonic.
[0306] Alternatively the composition may be formulated for topical
application for example in the form of ointments, creams, lotions,
eye ointments, eye drops, ear drops, mouthwash, impregnated
dressings and sutures and aerosols, and may contain appropriate
conventional additives, including, for example, preservatives,
solvents to assist drug penetration, and emollients in ointments
and creams. Such topical formulations may also contain compatible
conventional carriers, for example cream or ointment bases, and
ethanol or oleyl alcohol for lotions. Such carriers may constitute
from about 1% to about 98% by weight of the formulation; more
usually they will constitute up to about 80% by weight of the
formulation. Alternative means for systemic administration include
transmucosal and transdermal administration using penetrants such
as bile salts or fusidic acids or other detergents. In addition, if
a polypeptide or other compounds of the present invention can be
formulated in an enteric or an encapsulated formulation, oral
administration may also be possible. Administration of these
compounds may also be topical and/or localized, in the form of
salves, pastes, gels, and the like.
[0307] For administration to mammals, and particularly humans, it
is expected that the daily dosage level of the active agent will be
from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The
physician in any event will determine the actual dosage that will
be most suitable for an individual and will vary with the age,
weight and response of the particular individual. The above dosages
are exemplary of the average case. There can, of course, be
individual instances where higher or lower dosage ranges are
merited, and such are within the scope of this invention.
[0308] As used herein, the term "in-dwelling device" refers to
surgical implants, prosthetic devices and catheters, i.e., devices
that are introduced to the body of an individual and remain in
position for an extended time. Such devices include, but are not
limited to, artificial joints, heart valves, pacemakers, vascular
grafts, vascular catheters, cerebrospinal fluid shunts, urinary
catheters, continuous ambulatory peritoneal dialysis (CAPD)
catheters.
[0309] The composition of the invention may be administered by
injection to achieve a systemic effect against relevant bacteria
shortly before insertion of an in-dwelling device. Treatment may be
continued after surgery during the in-body time of the device. In
addition, the composition could also be used to broaden
perioperative cover for any surgical technique to prevent bacterial
wound infections, especially S. aureus wound infections.
[0310] Many orthopedic surgeons consider that humans with
prosthetic joints should be considered for antibiotic prophylaxis
before dental treatment that could produce a bacteremia. Deep
infection is a serious complication sometimes leading to loss of
the prosthetic joint and is accompanied by significant morbidity
and mortality. It may therefore be possible to extend the use of
the active agent as a replacement for prophylactic antibiotics in
this situation.
[0311] In addition to the therapy described above, the compositions
of this invention may be used generally as a wound treatment agent
to prevent adhesion of bacteria to matrix proteins exposed in wound
tissue and for prophylactic use in dental treatment as an
alternative to, or in conjunction with, antibiotic prophylaxis.
[0312] Alternatively, the composition of the invention may be used
to bathe an indwelling device immediately before insertion. The
active agent will preferably be present at a concentration of 1
mg/ml to 10 mg/ml for bathing of wounds or indwelling devices.
[0313] A vaccine composition is conveniently in injectable form.
Conventional adjuvants may be employed to enhance the immune
response. A suitable unit dose for vaccination is 0.5-5 microgram
of antigen/kg, and such dose is preferably administered 1-3 times
and with an interval of 1-3 weeks. With the indicated dose range,
no adverse toxicological effects will be observed with the
compounds of the invention that would preclude their administration
to suitable individuals.
[0314] Sequence Databases, Sequences in a Tangible Medium, and
Algorithms
[0315] Polynucleotide and polypeptide sequences form a valuable
information resource with which to determine their 2- and
3-dimensional structures as well as to identify further sequences
of homology. These approaches are most easily facilitated by
storing the sequence in a computer readable medium and then using
the stored data in a known macromolecular structure program or to
search a sequence database using well-known searching tools, such
as GCC.
[0316] The polynucleotide and polypeptide sequences of the
invention are particularly useful as components in databases useful
for search analyses as well as in sequence analysis algorithms. As
used in this section entitled "Sequence Databases, Sequences in a
Tangible Medium, and Algorithms," and in claims related to this
section, the terms "polynucleotide of the invention" and
"polynucleotide sequence of the invention" mean any detectable
chemical or physical characteristic of a polynucleotide of the
invention that is or may be reduced to or stored in a tangible
medium, preferably a computer readable form. For example,
chromatographic scan data or peak data, photographic data or scan
data therefrom, called bases, and mass spectrographic data. As used
in this section entitled Databases and Algorithms and in claims
related thereto, the terms "polypeptide of the invention" and
"polypeptide sequence of the invention" mean any detectable
chemical or physical characteristic of a polypeptide of the
invention that is or may be reduced to or stored in a tangible
medium, preferably a computer readable form. For example,
chromatographic scan data or peak data, photographic data or scan
data therefrom, and mass spectrographic data.
[0317] The invention provides a computer readable medium having
stored thereon polypeptide sequences of the invention and/or
polynucleotide sequences of the invention. The computer readable
medium can be any composition of matter used to store information
or data, including, for example, commercially available floppy
disks, tapes, chips, hard drives, compact disks, and video
disks.
[0318] In a preferred embodiment of the invention there is provided
a computer readable medium having stored thereon a member selected
from the group consisting of: a polynucleotide comprising the
sequence of SEQ ID NO: 1; a polypeptide comprising the sequence of
SEQ ID NO: 2; a set of polynucleotide sequences wherein at least
one of said sequences comprises the sequence of SEQ ID NO: 1: a set
of polypeptide sequences wherein at least one of said sequences
comprises the sequence of SEQ ID NO: 2; a data set representing a
polynucleotide sequence comprising the sequence of SEQ ID NO: 1; a
data set representing a polynucleotide sequence encoding a
polypeptide sequence comprising the sequence of SEQ ID NO: 2; a
polynucleotide comprising the sequence of SEQ ID NO: 1; a
polypeptide comprising the sequence of SEQ ID NO: 2; a set of
polynucleotide sequences wherein at least one of said sequences
comprises the sequence of SEQ ID NO: 1; a set of polypeptide
sequences wherein at least one of said sequences comprises the
sequence of SEQ ID NO: 2; a data set representing a polynucleotide
sequence comprising the sequence of SEQ ID NO: 1; a data set
representing a polynucleotide sequence encoding a polypeptide
sequence comprising the sequence of SEQ ID NO: 2.
[0319] All publications and references, including but not limited
to patents and patent applications, cited in this specification are
herein incorporated by reference in their entirety as if each
individual publication or reference were specifically and
individually indicated to be incorporated by reference herein as
being fully set forth. Any patent application to which this
application claims priority is also incorporated by reference
herein in its entirety in the manner described above for
publications and references.
[0320] The present invention is illustrated in further detail by
the following non-limiting examples.
EXAMPLE 1
[0321] Cloning of Inhibitory ORFs from Bacteriophage Genomes
[0322] We have used the methodology of a previous invention (PCT
International Application WO1999/IB99/02040, filed Dec. 3, 1999) to
identify inhibitory ORFs from bacteriophage 44AHJD and Twort. The
Staphylococcus aureus propagating strain (PS 44A) (Felix d'Herelle
Reference Centre #HER 1101, Ottawa, Canada) was used as a host to
propagate its respective phage 44AHJD (Felix d'Herelle Reference
Centre #HER101). The Staphylococcus propagating strain (PS Twort)
obtained from the Felix d'Herelle Reference Centre maintained by Dr
H.-W. Ackermann (Quebec, Canada) (#HER 1048) was used as a host to
propagate the phage Twort, also obtained from the Felix d'Herelle
Reference Centre (#HER 48).
[0323] The Staphylococcus aureus propagating strain PS15 (ATCC
27712), obtained from American Type Culture Collection (Manassas,
Va., USA) was used as a host to propagate phage G1. Phage G1 was
isolated from a cocktail of S. aureus phages (Bacteriophagum
staphylococcum liquidum, lot number 361098) manufactured by
BioPharm, Tblisi, Republic of Georgia, by infection of PS15. The
cocktail of Staphylococcus aureus phages was purchased from a
drugstore in Tblisi.
[0324] The inhibitory ORF corresponding to SEQ ID NO: 4, 6 and 10
were amplified by polymerase chain reaction (PCR) from genomic DNA
derived from phage 44AHJD, Twort and G1 respectively. As
examplified in FIG. 3A for 44AHJD ORF 25 and Twort ORF 168, the PCR
products were cloned into pT and pTM, two modified versions of the
pT0021, a sodium arsenite inducible expression vector [Tauriainen
et al., 1997, Appl. Environ. Microbiol. 63:4456-4461]. As shown in
FIG. 3B, the density of the culture, as assessed by colony forming
units (CFU), for S. aureus clones harboring inhibitory ORFs
increased over time under non-induced conditions. Similar growth
rates were also observed with transformants harboring a
non-inhibitory ORF (labeled as `non killer` on the graphs) under
both induced and non-induced conditions. Each graph represents the
average obtained from three independent transformants of S. aureus.
The expression of 44AHJD ORF 25 and Twort ORF168 inhibit the
bacterial growth as observed by the reduction in CFU with time for
induced cultures (5.0 uM sodium arsenite). The expression of G1 ORF
240 similarly inhibited the growth of S. aureus (results not
shown).
EXAMPLE 2
Identification of a S. Aureus Protein Targeted by Bacteriophage
44AHJD ORF 25
[0325] A. Generation of GST/ORF 25 Recombinant Protein
[0326] Bacteriophage 44AHJD ORF 25 was sub-cloned into pGEX 4T-1
(Pharmacia), an expression vector containing the GST moiety. ORF 25
was obtained by digestion of pT/44AHJD ORF 25 with BamHI and SalI.
The DNA fragment containing ORF 25 was gel purified by QiaQuick.TM.
spin columns (Qiagen) and ligated into pGEX 4T-1 (which had been
previously digested with BamHI and SalI) to generate pGEX 4T
GST/ORF 25. Recombinant expression vectors were identified by
restriction enzyme analysis of plasmid minipreps. Large-scale DNA
preparations were performed and the resulting insert was sequenced.
Test expressions in E. coli BL21 (DE3) Gold cells containing the
expression plasmids were performed to identify optimal protein
expression conditions. E. coli cells containing the expression
constructs were grown in Luria-Bertani Broth at 25.degree. C. to an
OD.sub.600 of 0.4 to 0.6 and induced with 1 mM IPTG for the optimal
times and at the optimal temperatures (typically a 2 liter culture
of BL21 (DE3) Gold (pGEX 4T/ORF25) grown at 25.degree. C. for 3
hrs).
[0327] B. Fusion Protein Purification.
[0328] Cells containing GST/ORF 25 fusion protein were suspended in
15 ml lysis buffer/liter of cell culture with GST lysis buffer (20
mM Hepes pH 7.2, 500 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA, 1
mM benzamidine, and 1 PMSF) and lysed using a French pressure cell
followed by three bursts of twenty seconds with an ultra-sonicator
at 4.degree. C. Triton X-100 was added to the lysate to a final
concentration of 0.1% and mixed for 30 minutes at 4.degree. C. The
lysate was centrifuged at 4.degree. C. for 30 minutes at 10,000 rpm
in a Sorval SS34 rotor. The supernatant was applied to a 4 ml
glutathione sepharose column pre-equilibrated with lysis buffer and
allowed to flow by gravity. The column was washed with 10 column
volumes of lysis buffer and eluted in 1.5 ml fractions with GST
elution buffer (20 mM Hepes pH 8.0, 500 mM NaCl, 10% glycerol, 1 mM
DTT, 0.1 mM EDTA, and 25 mM reduced glutathione). The fractions
were analyzed by SDS-12.5% PAGE (Laemmli) and proteins were
visualized by staining with Coomassie Brilliant Blue R250 stain to
assess the amount of eluted GST/ORF 25 protein.
[0329] C. Affinity Column Preparation.
[0330] GST and GST/ORF25 were dialyzed overnight against affinity
chromatography buffer (ACB; 20 mM Hepes pH 7.5, 10% glycerol, 1 mM
DTT, and 1 mM EDTA) containing 1 M NaCl. Protein concentrations
were determined by Bio-Rad Protein Assay and crosslinked to Affigel
10 resin (Bio-Rad) at protein/resin concentrations of 0, 0.1, 0.5,
1.0, and 2.0 mg/ml. The crosslinked resin was sequentially
incubated in the presence of ethanolamine, and bovine serum albumin
(BSA) prior to column packing and equilibration with ACB containing
100 mM NaCl.
[0331] D. S. aureus Extract Preparation.
[0332] Two extracts were prepared from S. aureus cell pellets. One
lysate was prepared by French pressure cell lysis followed by
sonication, and the other by lysostaphin-mediated digestion
followed by sonication. The French pressure cell lysate was
prepared by suspending 3 g of frozen S. aureus cells in ABC
containing 500 mM NaCl, 1 mM PMSF, and 1 mM benzamidine. The
suspended cells were subjected to three passes through the French
pressure cell followed by 3 sonication bursts of 20 seconds each,
made up to 0.1% Triton X-100, stirred for 30 minutes, and
centrifuged at 50,000 rpm for 3 hrs in a Ti70 fixed angle Beckman
rotor. The efficiency of cell lysis was low and the resulting
lysate (7 ml) contained 2.4 mg/ml protein. The pellet after French
pressure cell lysis was subjected to cryogenic grinding in liquid
nitrogen in the same buffer with a mortar and pestle. The lysate
was made up to 0.1% Triton X-100, stirred for 30 minutes, and
centrifuged at 50,000 rpm for 3 hrs in a Ti70 fixed angle Beckman
rotor yielding a lysate (10 ml) containing 2.0 mg/ml protein. The
cell lysates were pooled, concentrated to 8 ml, and dialyzed
overnight in a 3000 Mr cut-off dialysis membrane against ACB
containing 1 mM PMSF, 1 mM benzamidine, and 75 mM NaCl. The
dialyzed protein extract was removed from the dialysis tubing,
centrifuged at 10 000 rpm in a Sorval SS34 rotor for 1 hr, and
assayed for protein content (Bio-Rad Protein Assay) and salt
concentration (conductivity meter).
[0333] E. Affinity Chromatography.
[0334] Affinity chromatography was performed using GST and GST
ORF25 as ligands coupled to Affigel 10 at protein/resin
concentrations of 0, 0.1, 0.5, 1.0, and 2.0 mg/ml. The S. aureus
extract was centrifuged at 4.degree. C. in a micro-centrifuge for
15 minutes and 200 ul was applied to 20 ul columns containing 0,
0.1, 0.5, 1.0, and 2.0 mg/ml ligand. ACB containing 100 mM NaCl
(200 ul) was applied to a control column containing 2.0 mg/ml
ligand. The columns were washed with 10 column volumes ACB
containing 100 mM NaCl and sequentially eluted with ACB containing
1% Triton X-100 and 100 mM NaCl (800 ul), ACB containing 1 M NaCl
(800 ul), and 1% SDS (160 ul). 40 ul of each eluate was resolved by
SDS-12.5% PAGE (Laemmli) and the protein was visualized by silver
stain.
[0335] F. Identification of S. Aureus DnaN as an 44AHJD ORF 25
Interacting Protein
[0336] Two S. aureus extracts were used for affinity chromatography
with each of the ligands. Two extracts used for affinity
chromatography, prepared separately, contained 4.0 and 9.0 mg/ml
protein. One candidate interacting protein of 48 kDa (PT48) was
observed in the 1% SDS eluates in the initial chromatography
experiment (FIG. 4). The candidate protein, PT48 was excised from
the SDS-PAGE gels and prepared for tryptic peptide mass
determination by MALDI-ToF mass spectrometry [Qin, J., Fenyo, D.,
Zhao, Y., Hall, W. W., Chao, D. M., Wilson, C. J., Young, R. A. and
Chait, B. T. (1997) Anal. Chem. 69, 3995-4001]. High quality mass
spectra were obtained (FIG. 5). The PT48 proteins observed in two
affinity chromatography experiments were identical as determined by
the masses of the tryptic peptides. Computational analysis
(http://prowl.rockfeller.edu- /cgi-bin/ProFound) of the mass
spectrum obtained identifies the corresponding ORF in the S. aureus
nucleotide sequence in the University of Oklahoma S. aureus genomic
database (http://www.genome.ou.edu/staph.ht- ml). The identity of
that protein which binds specifically to GST ORF25 is the
DNA-directed DNA polymerase III beta subunit (Genbank accession
#1084187) (FIG. 6).
[0337] The identification of S. aureus STAAU_R2 as an interacting
partner of bacteriophage 44ADJH ORF 25 was also validated by
surface plasmon resonance (Biacore 2000 Biosensor) using purified
recombinant polypeptides. Glutathione-S-transferase (GST)-tagged
STAAU_R2 was captured as ligand by an anti-GST antibody which had
been covalently coupled to the surface of a CM5 sensor chip; a
blank surface with anti-GST antibody and without captured ligand
was used as a negative control. Injection of purified 44ADJH ORF 25
protein over the two surfaces indicated specific capture of 44ADJH
ORF 25 by immobilized STAAU_R2.
EXAMPLE 3
Identification of a S. Aureus Protein Targeted by Bacteriophage
Twort ORF168
[0338] A. Generation of GST/Twort ORF168 Recombinant Protein
[0339] Bacteriophage Twort ORF 168 was sub-cloned into pGEX 4T-1.
Plasmid (pTM) containing Twort ORF 168 was purified on a Qiagen
column and digested with HindIII, treated with Klenow fragment of E
coli DNA polymerase, and digested with BamHI. The DNA restriction
products containing the ORF was gel purified by QiAquick spin
column (Qiagen) and ligated into pGEX 4T-1 expression vector
(prepared by digestion with SalI, treatment with Klenow fragment of
E. coli DNA polymerase, and digestion with BamH1, followed by gel
purification by QiAquick spin columns). Recombinant expression
vectors were identified by restriction enzyme analysis of plasmid
minipreps, and large-scale DNA preparations were performed with
Qiagen DNA purification columns. Test expressions in E. coli cells
containing the expression plasmids were performed to identify
optimal protein expression conditions (expressed in BL21(DE3) Gold
cells). E. coli cells containing the expression constructs were
grown in Luria-Bertani Broth at 37.degree. C. to an OD.sub.600 of
0.4 to 0.6 and induced with 1 mM IPTG for the optimal times and at
the optimal temperatures (2 liters of GST ORF168 at 15.degree. C.
for 16 hrs).
[0340] B. Fusion Protein Purification.
[0341] Cells containing GST ORF 168 fusion protein were suspended
in 10 ml lysis buffer/liter of cell culture with GST lysis buffer
and lysed by three bursts of twenty seconds with an ultra-sonicator
at 4.degree. C. The lysate was centrifuged at 4.degree. C. for 30
minutes at 10 000 rpm in a Beckman JA25.50 rotor. The supernatant
was applied to a 4 ml glutathione sepharose column pre-equilibrated
with lysis buffer and allowed to flow by gravity. The column was
washed with 10 column volumes of lysis buffer and eluted in 4 ml
fractions with GST elution buffer. The fractions were analyzed by
15% SDS-PAGE (Laemmli) and visualized by staining with Coomassie
Brilliant Blue R250 stain.
[0342] C. Affinity Column Preparation.
[0343] GST, and GST ORF168 were dialyzed overnight against ACB at
pH 7.9 containing 1 M NaCl. Protein concentrations were determined
by Bio-Rad Protein Assay and crosslinked to Affigel 10 resin
(Bio-Rad) at protein/resin concentrations of 0, 0.1, 0.5, 1.0, and
2.0 mg/ml. The resin containing crosslinked protein was washed with
ACB containing 1M NaCl and equilibrated with ACB containing 100 mM
NaCl.
[0344] D. S. aureus Extract Preparation.
[0345] Staphylococcus aureus extract was prepared from a cell
pellet using a bead beater lysis method and nuclease treatment. A
Staphylococcus aureus cell pellet (2.9 g) was suspended in 8 ml of
20 mM Hepes pH 7.5, 500 mM NaCl, 10% glycerol, 10 mM MgSO.sub.4, 10
mM CaCl.sub.2, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine, 0.5 mg RNAse
A, and 750 units micrococcal nuclease. The cell suspension was
added to 15 ml of zirconia/silica glass beads (0.1 mm diameter) at
4.degree. C., and subjected to bead beater pulses (30 seconds on,
90 seconds off, using a super-cooled ice bath at -18.degree. C.).
The beads were separated from the lysate with a Biorad Econo column
(2.5.times.40 cm) and the beads washed with one column volume of
lysis buffer. The flow through and the wash were pooled and
centrifuged at 20 000 rpm for 1 hrs in a Beckman JA25.50 rotor. The
supernatant was removed and dialyzed overnight in a 10 000 Mr
dialysis membrane against ACB (20 mM Hepes pH 7.5, 10% glycerol, 1
mM DTT, and 1 mM EDTA) containing 100 mM NaCl, 1 mM benzamidine, 10
mM MgSO.sub.4, 10 mM CaCl.sub.2, and 1 mM PMSF. The dialyzed
protein extract was removed from the dialysis tubing and frozen in
one ml aliquots at -70.degree. C.
[0346] E. Affinity Chromatography
[0347] Affinity chromatography was performed using GST and GST
ORF168 as ligands coupled to Affigel 10 at protein/resin
concentrations of 0, 0.1, 0.5, 1.0, and 2.0 mg/ml. Staphylococcus
aureus extracts were centrifuged at 4.degree. C. in a
micro-centrifuge for 15 minutes and diluted to 5 mg/ml with ACB
containing 100 mM NaCl. 200 .mu.l of extract was applied to 40
.mu.l columns containing 0, 0.1, 0.5, 1.0, and 2.0 mg/ml ligand and
ACB containing 100 mM NaCl (200 .mu.l) was applied to an additional
column containing 2.0 mg/ml ligand. The columns were washed twice
with ACB containing 100 mM NaCl (2.times.100 .mu.l), ACB containing
0.1% Triton X-100 and 100 mM NaCl (200 .mu.l), and sequentially
eluted with ACB containing 1 M NaCl (160 .mu.l), and 1% SDS (160
.mu.l). 65 .mu.l of each eluate was resolved by 16 cm 14% SDS-PAGE
(Laemmli) and the protein was visualized by silver stain.
[0348] F. Identification of S. Aureus DnaN as a Twort ORF 168
Interacting Protein
[0349] One candidate interacting protein of 50 kDa (PT50) was
observed to interact with ORF 168. PT50 was observed in both the 1
M NaCl eluates, and the 1% SDS eluates of the GST ORF168
chromatography experiment (FIG. 7A). Approximately 10% of the PT50
was observed in the 1 M NaCl eluates while 90% was observed in the
1% SDS eluates. PT50 was not observed in the GST control affinity
chromatography experiment (FIG. 7B).
[0350] High quality mass spectra were obtained (FIG. 8). The PT50
proteins observed in affinity chromatography experiments were
identical as determined by the masses of the tryptic peptides. The
identity of that protein which binds specifically to GST ORF 168 is
the DNA-directed DNA polymerase III beta subunit (Genbank accession
#1084187) (FIG. 9).
EXAMPLE 4
Confirmation of the Interaction between STAAU_R2 and Twort ORF168
by Yeast Two-Hybrid Analysis
[0351] To validate the identification of S. aureus STAAU_R2 as an
interacting partner of bacteriophage Twort ORF 168 and to identify
a specific domain of Twort ORF 168 which participates in the
interaction with S. aureus STAAU_R2, recombinant Twort ORF 168
protein was subjected to deletion analysis using the yeast
two-hybrid system.
[0352] A. Generation of Twort ORF 168 and STAAU_R2 Recombinant
Polypeptides for Yeast Two-Hybrid Analysis.
[0353] Bacteriophage Twort ORF 168 was fused either to the carboxyl
terminus of the yeast Gal4 DNA binding domain (encoded in the
pGBKT7 vector from Clontech Laboratories) or to the yeast Gal4
activation domain (encoded in the pGADT7 vector from Clontech
Laboratories). As illustrated in FIG. 10B, the sense strand primer
(SEQ ID NO: 11; 5-ccggaattcATGTTATTTTTTAAAGAAAAG-3') is preceded by
a EcoRI restriction site; the antisense oligonucleotide (SEQ ID NO:
12; 5'-cgcggatccTCATCGMCTATATCCTTAAT-3') targets the stop codon and
is preceded by a BamHI restriction site. The PCR product was
purified using the Qiagen PCR purification kit and digested with
EcoRI and BamHI. The digested PCR product was ligated to EcoRI- and
BamHI-digested pGBKT7 vector, yielding pGBKSTAAU_R2. A similar
strategy was used for the cloning of STAAU_R2 into the pGADT7
vector yielding pGADSTAAU_R2.
[0354] Fragment of Twort ORF 168 extending from amino acid residues
5 to 40 was amplified by PCR and cloned using the same strategy.
The sense strand primer (SEQ ID NO: 13;
5-ccggaattcAAAGAAAAGTTTTATAATGAAT-3') targets the initiation codon
and is preceded by a EcoRI restriction site; the antisense
oligonucleotide (SEQ ID NO: 14; 5'-cgcggatccTCAATCTTCTTCTTC-
TAATTTCTC-3') targets the stop codon and is preceded by a BamHI
restriction site.
[0355] The polynucleotide sequence of STAAU_R2 was obtained from S.
aureus genomic DNA by PCR utilizing oligonucleotide primers that
targeted the predicted translation initiation and termination
codons of the STAAU_R2 gene (SEQ ID NO: 1).
[0356] As illustrated in FIG. 10A, the sense strand primer (SEQ ID
NO: 15; 5-GGGAATTCCATATGATGATGGAATTCACTATTAAA-3') targets the
initiation codon and is preceded by a NdeI restriction site; the
antisense oligonucleotide (SEQ ID NO: 16; 5'-CGCGGATCC
TTAGTAAGTTCTGATTGG-3') targets the stop codon and is preceded by a
BamHI restriction site. The PCR product was purified using the
Qiagen PCR purification kit and digested with NdeI and BamHI. The
digested PCR product was ligated to NdeI- and BamHI-digested pGADT7
vector (Clontech Laboratories), yielding pGADSTAAU_R2. A similar
strategy was used for the cloning of STAAU_R2 into the pGBKT7
vector (Clontech Laboratories), yielding pGBKSTAAU_R2.
[0357] B. Yeast Two-Hybrid Analysis
[0358] As shown in FIGS. 11A and B, the pGAD and pGBK plasmids
bearing different combinations of constructs (as indicated above
each pair of petri plates) were introduced into a yeast strain
(AH109, Clontech Laboratories), previously engineered to contain
chromosomally-integrated copies of E. coli lacZ and the selectable
HIS3 and ADE2 genes. Co-transformants were plated in parallel on
yeast synthetic medium (SD) supplemented with amino acid drop-out
lacking tryptophan and leucine (TL minus) and on SD supplemented
with amino acid drop-out lacking tryptophan, histidine, adenine and
leucine (THAL minus). Co-transformants harbouring the Twort ORF 168
polypeptide only grew on selective SD THAL minus medium in the
presence of STAAU_R2. Induction of the reporter HIS3 and ADE2 genes
is dependent upon the interaction of STAAU_R2 with Twort ORF 168
since cotransformants with appropriate control plasmids
(pGBKT7LaminC or pGADT7-T) are not viable on SD THAL minus
medium.
[0359] The interaction of STAAU_R2 and Twort ORF 168 was also
clearly demonstrated by the observed increase, over the background
level, of the .beta.-galactosidase activity in both Twort ORF
168-STAAU_R2 co-transformants (FIG. 11C samples 1 and 4
respectively). These results are consistent with the interpretation
that the S. aureus STAAU_R2 identified herein is the host target of
bacteriophage Twort ORF 168.
[0360] The interaction of STAAU_R2 and a Twort ORF 168-related
fragment was also demonstrated (results not shown). A portion of 36
amino acid sequence of Twort ORF 168 extending from amino acids
residues 5 to 40 (herein referred to as SEQ ID NO: 8) was found to
interact with STAAU_R2 since the introduction of appropriate
plasmids into host yeast cells resulted in their growth on THAL
minus SD medium.
EXAMPLE 5
Identification of STAAU_R2 as Targeted by Bacteriophage G1 ORF
240
[0361] A. Generation of GST/G1 ORF 240 Recombinant Protein
[0362] As shown in FIG. 12, the optimal global aligment of G1 ORF
240 and Twort ORF 168 reveals an identity of 29% between the two
polypeptides. The optimal local aligment of G1 ORF 240 shows a 47%
identity to the polypeptide corresponding to SEQ ID NO: 8.
[0363] G1 ORF 240 was cloned into pGEX 4T-1 and the polypeptide was
purified as described above. Affinity chromatography was performed
using GST and GST ORF240 as ligands coupled to Affigel 10 at
protein/resin concentrations of 0, 0.1, 0.5, 1.0, and 2.0 mg/ml.
Staphylococcus aureus extracts was applied to columns and ACB
containing 100 mM NaCl was applied to an additional column
containing 2.0 mg/ml ligand. The columns were washed and
sequentially eluted with ACB containing 1 M NaCl, and 1% SDS. A
fraction of each eluate was resolved by 14% SDS-PAGE and the
protein was visualized by silver stain.
[0364] One candidate interacting protein was observed to
specifically interact with ORF 240 and was shown to correspond to
STAAU_R2 (results not shown).
CONCLUSION
[0365] By virtue of the interaction between the inhibitory
bacteriophage 44AHJD ORF 25, Twort ORF 168, G1 ORF 240_and the
STAAU_R2, the STAAU_R2 gene and its gene product have thus been
identified as novel bacterial targets for the screening and
identification of anti-bacterial agents and more particularly for
anti S. aureus agents. The present invention also provides novel
diagnosis, prognosis and therapeutic methods based on STAAU_R2,
and/or bacteriophage 44AHJD ORF 25 and/or Twort ORF 168, G1 ORF 240
and/or a compound identified in accordance with the present
invention.
[0366] Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be
modified without departing from the spirit and nature of the
subject invention as defined in the appended claims.
Sequence CWU 1
1
16 1 1134 DNA Staphylococcus aureus CDS (1)..(1134) 1 atg atg gaa
ttc act att aaa aga gat tat ttt att aca caa tta aat 48 Met Met Glu
Phe Thr Ile Lys Arg Asp Tyr Phe Ile Thr Gln Leu Asn 1 5 10 15 gac
aca tta aaa gct att tca cca aga aca aca tta cct ata tta act 96 Asp
Thr Leu Lys Ala Ile Ser Pro Arg Thr Thr Leu Pro Ile Leu Thr 20 25
30 ggt atc aaa atc gat gcg aaa gaa cat gaa gtt ata tta act ggt tca
144 Gly Ile Lys Ile Asp Ala Lys Glu His Glu Val Ile Leu Thr Gly Ser
35 40 45 gac tct gaa att tca ata gaa atc act att cct aaa act gta
gat ggc 192 Asp Ser Glu Ile Ser Ile Glu Ile Thr Ile Pro Lys Thr Val
Asp Gly 50 55 60 gaa gat att gtc aat att tca gaa aca ggc tca gta
gta ctt cct gga 240 Glu Asp Ile Val Asn Ile Ser Glu Thr Gly Ser Val
Val Leu Pro Gly 65 70 75 80 cga ttc ttt gtt gat att ata aaa aaa tta
cct ggt aaa gat gtt aaa 288 Arg Phe Phe Val Asp Ile Ile Lys Lys Leu
Pro Gly Lys Asp Val Lys 85 90 95 tta tct aca aat gaa caa ttc cag
aca tta att aca tca ggt cat tct 336 Leu Ser Thr Asn Glu Gln Phe Gln
Thr Leu Ile Thr Ser Gly His Ser 100 105 110 gaa ttt aat tta agt ggc
tta gat cca gat caa tat cct tta tta cct 384 Glu Phe Asn Leu Ser Gly
Leu Asp Pro Asp Gln Tyr Pro Leu Leu Pro 115 120 125 caa gtt tct aga
gat gac gca att caa ttg tcg gta aaa gtg ctt aaa 432 Gln Val Ser Arg
Asp Asp Ala Ile Gln Leu Ser Val Lys Val Leu Lys 130 135 140 aac gtg
att gca caa aca aat ttt gca gtg tcc acc tca gaa aca cgc 480 Asn Val
Ile Ala Gln Thr Asn Phe Ala Val Ser Thr Ser Glu Thr Arg 145 150 155
160 cca gta cta act ggt gtg aac tgg ctt ata caa gaa aat gaa tta ata
528 Pro Val Leu Thr Gly Val Asn Trp Leu Ile Gln Glu Asn Glu Leu Ile
165 170 175 tgc aca gcg act gac tca cac cgc ttg gct gta aga aag ttg
cag tta 576 Cys Thr Ala Thr Asp Ser His Arg Leu Ala Val Arg Lys Leu
Gln Leu 180 185 190 gaa gat gtt tct gaa aac aaa aat gtc atc att cca
ggt aag gct tta 624 Glu Asp Val Ser Glu Asn Lys Asn Val Ile Ile Pro
Gly Lys Ala Leu 195 200 205 gct gaa tta aat aaa att atg tct gac aat
gaa gaa gac att gat atc 672 Ala Glu Leu Asn Lys Ile Met Ser Asp Asn
Glu Glu Asp Ile Asp Ile 210 215 220 ttc ttt gct tca aac caa gtt tta
ttt aaa gtt gga aat gtg aac ttt 720 Phe Phe Ala Ser Asn Gln Val Leu
Phe Lys Val Gly Asn Val Asn Phe 225 230 235 240 att tct cga tta tta
gaa gga cat tat cct gat aca aca cgt tta ttc 768 Ile Ser Arg Leu Leu
Glu Gly His Tyr Pro Asp Thr Thr Arg Leu Phe 245 250 255 cct gaa aac
tat gaa att aaa tta agt ata gac aat ggg gag ttt tat 816 Pro Glu Asn
Tyr Glu Ile Lys Leu Ser Ile Asp Asn Gly Glu Phe Tyr 260 265 270 cat
gcg att gat cgt gcc tct tta tta gcg cgt gaa ggt ggt aat aac 864 His
Ala Ile Asp Arg Ala Ser Leu Leu Ala Arg Glu Gly Gly Asn Asn 275 280
285 gtt att aaa tta agt aca ggt gat gac gtt gtt gaa ttg tct tct aca
912 Val Ile Lys Leu Ser Thr Gly Asp Asp Val Val Glu Leu Ser Ser Thr
290 295 300 tca cca gaa att ggt act gta aaa gaa gaa gtt gat gca aac
gat gtt 960 Ser Pro Glu Ile Gly Thr Val Lys Glu Glu Val Asp Ala Asn
Asp Val 305 310 315 320 gaa ggt ggt agc ctg aaa att tca ttc aac tct
aaa tat atg atg gat 1008 Glu Gly Gly Ser Leu Lys Ile Ser Phe Asn
Ser Lys Tyr Met Met Asp 325 330 335 gct tta aaa gca atc gat aat gat
gag gtt gaa gtt gaa ttc ttc ggt 1056 Ala Leu Lys Ala Ile Asp Asn
Asp Glu Val Glu Val Glu Phe Phe Gly 340 345 350 aca atg aaa cca ttt
att cta aaa cca aaa ggt gac gac tcg gta acg 1104 Thr Met Lys Pro
Phe Ile Leu Lys Pro Lys Gly Asp Asp Ser Val Thr 355 360 365 caa tta
att tta cca atc aga act tac taa 1134 Gln Leu Ile Leu Pro Ile Arg
Thr Tyr 370 375 2 377 PRT Staphylococcus aureus 2 Met Met Glu Phe
Thr Ile Lys Arg Asp Tyr Phe Ile Thr Gln Leu Asn 1 5 10 15 Asp Thr
Leu Lys Ala Ile Ser Pro Arg Thr Thr Leu Pro Ile Leu Thr 20 25 30
Gly Ile Lys Ile Asp Ala Lys Glu His Glu Val Ile Leu Thr Gly Ser 35
40 45 Asp Ser Glu Ile Ser Ile Glu Ile Thr Ile Pro Lys Thr Val Asp
Gly 50 55 60 Glu Asp Ile Val Asn Ile Ser Glu Thr Gly Ser Val Val
Leu Pro Gly 65 70 75 80 Arg Phe Phe Val Asp Ile Ile Lys Lys Leu Pro
Gly Lys Asp Val Lys 85 90 95 Leu Ser Thr Asn Glu Gln Phe Gln Thr
Leu Ile Thr Ser Gly His Ser 100 105 110 Glu Phe Asn Leu Ser Gly Leu
Asp Pro Asp Gln Tyr Pro Leu Leu Pro 115 120 125 Gln Val Ser Arg Asp
Asp Ala Ile Gln Leu Ser Val Lys Val Leu Lys 130 135 140 Asn Val Ile
Ala Gln Thr Asn Phe Ala Val Ser Thr Ser Glu Thr Arg 145 150 155 160
Pro Val Leu Thr Gly Val Asn Trp Leu Ile Gln Glu Asn Glu Leu Ile 165
170 175 Cys Thr Ala Thr Asp Ser His Arg Leu Ala Val Arg Lys Leu Gln
Leu 180 185 190 Glu Asp Val Ser Glu Asn Lys Asn Val Ile Ile Pro Gly
Lys Ala Leu 195 200 205 Ala Glu Leu Asn Lys Ile Met Ser Asp Asn Glu
Glu Asp Ile Asp Ile 210 215 220 Phe Phe Ala Ser Asn Gln Val Leu Phe
Lys Val Gly Asn Val Asn Phe 225 230 235 240 Ile Ser Arg Leu Leu Glu
Gly His Tyr Pro Asp Thr Thr Arg Leu Phe 245 250 255 Pro Glu Asn Tyr
Glu Ile Lys Leu Ser Ile Asp Asn Gly Glu Phe Tyr 260 265 270 His Ala
Ile Asp Arg Ala Ser Leu Leu Ala Arg Glu Gly Gly Asn Asn 275 280 285
Val Ile Lys Leu Ser Thr Gly Asp Asp Val Val Glu Leu Ser Ser Thr 290
295 300 Ser Pro Glu Ile Gly Thr Val Lys Glu Glu Val Asp Ala Asn Asp
Val 305 310 315 320 Glu Gly Gly Ser Leu Lys Ile Ser Phe Asn Ser Lys
Tyr Met Met Asp 325 330 335 Ala Leu Lys Ala Ile Asp Asn Asp Glu Val
Glu Val Glu Phe Phe Gly 340 345 350 Thr Met Lys Pro Phe Ile Leu Lys
Pro Lys Gly Asp Asp Ser Val Thr 355 360 365 Gln Leu Ile Leu Pro Ile
Arg Thr Tyr 370 375 3 177 DNA Bacteriophage 44AHJD CDS (1)..(177) 3
atg gaa cgt aaa tac aaa acg gta tta tta tat tgc gat gag att aaa 48
Met Glu Arg Lys Tyr Lys Thr Val Leu Leu Tyr Cys Asp Glu Ile Lys 1 5
10 15 gga cat ttt cca cat caa atc tca atg ttt gaa gat tta tat gac
gct 96 Gly His Phe Pro His Gln Ile Ser Met Phe Glu Asp Leu Tyr Asp
Ala 20 25 30 aaa gtt gta tat tca tat tat gaa tat aac ctg ttc act
aaa aaa tac 144 Lys Val Val Tyr Ser Tyr Tyr Glu Tyr Asn Leu Phe Thr
Lys Lys Tyr 35 40 45 gcg tat atc ata gaa tac att aag gag ata taa
177 Ala Tyr Ile Ile Glu Tyr Ile Lys Glu Ile 50 55 4 58 PRT
Bacteriophage 44AHJD 4 Met Glu Arg Lys Tyr Lys Thr Val Leu Leu Tyr
Cys Asp Glu Ile Lys 1 5 10 15 Gly His Phe Pro His Gln Ile Ser Met
Phe Glu Asp Leu Tyr Asp Ala 20 25 30 Lys Val Val Tyr Ser Tyr Tyr
Glu Tyr Asn Leu Phe Thr Lys Lys Tyr 35 40 45 Ala Tyr Ile Ile Glu
Tyr Ile Lys Glu Ile 50 55 5 225 DNA Bacteriophage Twort CDS
(1)..(225) 5 atg tta ttt ttt aaa gaa aag ttt tat aat gaa tta agt
tat tat aga 48 Met Leu Phe Phe Lys Glu Lys Phe Tyr Asn Glu Leu Ser
Tyr Tyr Arg 1 5 10 15 ggt gga cac aag gat tta gaa agt atg ttt gag
tta gcg tta gag tat 96 Gly Gly His Lys Asp Leu Glu Ser Met Phe Glu
Leu Ala Leu Glu Tyr 20 25 30 att gag aaa tta gaa gaa gaa gat gaa
cag cag gta act gat tat gag 144 Ile Glu Lys Leu Glu Glu Glu Asp Glu
Gln Gln Val Thr Asp Tyr Glu 35 40 45 aac gct atg gaa gaa gaa tta
agg gat gct gtt gat gta att gag agt 192 Asn Ala Met Glu Glu Glu Leu
Arg Asp Ala Val Asp Val Ile Glu Ser 50 55 60 cag tta gaa att att
aag gat ata gtt cga tga 225 Gln Leu Glu Ile Ile Lys Asp Ile Val Arg
65 70 6 74 PRT Bacteriophage Twort 6 Met Leu Phe Phe Lys Glu Lys
Phe Tyr Asn Glu Leu Ser Tyr Tyr Arg 1 5 10 15 Gly Gly His Lys Asp
Leu Glu Ser Met Phe Glu Leu Ala Leu Glu Tyr 20 25 30 Ile Glu Lys
Leu Glu Glu Glu Asp Glu Gln Gln Val Thr Asp Tyr Glu 35 40 45 Asn
Ala Met Glu Glu Glu Leu Arg Asp Ala Val Asp Val Ile Glu Ser 50 55
60 Gln Leu Glu Ile Ile Lys Asp Ile Val Arg 65 70 7 108 DNA
Bacteriophage Twort CDS (1)..(108) 7 aaa gaa aag ttt tat aat gaa
tta agt tat tat aga ggt gga cac aag 48 Lys Glu Lys Phe Tyr Asn Glu
Leu Ser Tyr Tyr Arg Gly Gly His Lys 1 5 10 15 gat tta gaa agt atg
ttt gag tta gcg tta gag tat att gag aaa tta 96 Asp Leu Glu Ser Met
Phe Glu Leu Ala Leu Glu Tyr Ile Glu Lys Leu 20 25 30 gaa gaa gaa
gat 108 Glu Glu Glu Asp 35 8 36 PRT Bacteriophage Twort 8 Lys Glu
Lys Phe Tyr Asn Glu Leu Ser Tyr Tyr Arg Gly Gly His Lys 1 5 10 15
Asp Leu Glu Ser Met Phe Glu Leu Ala Leu Glu Tyr Ile Glu Lys Leu 20
25 30 Glu Glu Glu Asp 35 9 177 DNA Bacteriophage G1 CDS (1)..(177)
9 atg gtt ata cct agt att aaa gca caa aac aaa ttc aag aat gag tta
48 Met Val Ile Pro Ser Ile Lys Ala Gln Asn Lys Phe Lys Asn Glu Leu
1 5 10 15 gag tat tat aaa caa ggt cac att agt gaa agt aaa atg tta
gaa tta 96 Glu Tyr Tyr Lys Gln Gly His Ile Ser Glu Ser Lys Met Leu
Glu Leu 20 25 30 gct ttt gat tac atc caa gaa tta gaa caa aat aac
gaa tac gtt act 144 Ala Phe Asp Tyr Ile Gln Glu Leu Glu Gln Asn Asn
Glu Tyr Val Thr 35 40 45 aac ttg cta gaa gag gag aga tat ggt gag
taa 177 Asn Leu Leu Glu Glu Glu Arg Tyr Gly Glu 50 55 10 58 PRT
Bacteriophage G1 10 Met Val Ile Pro Ser Ile Lys Ala Gln Asn Lys Phe
Lys Asn Glu Leu 1 5 10 15 Glu Tyr Tyr Lys Gln Gly His Ile Ser Glu
Ser Lys Met Leu Glu Leu 20 25 30 Ala Phe Asp Tyr Ile Gln Glu Leu
Glu Gln Asn Asn Glu Tyr Val Thr 35 40 45 Asn Leu Leu Glu Glu Glu
Arg Tyr Gly Glu 50 55 11 30 DNA artificial sequence Sequence is
completely synthesized 11 ccggaattca tgttattttt taaagaaaag 30 12 30
DNA artificial sequence Sequence is completely synthesized 12
cgcggatcct catcgaacta tatccttaat 30 13 31 DNA artificial sequence
Sequence is completely synthesized 13 ccggaattca aagaaaagtt
ttataatgaa t 31 14 33 DNA artificial sequence Sequence is
completely synthesized 14 cgcggatcct caatcttctt cttctaattt ctc 33
15 35 DNA artificial sequence Sequence is completely synthesized 15
gggaattcca tatgatgatg gaattcacta ttaaa 35 16 27 DNA artificial
sequence Sequence is completely synthesized 16 cgcggatcct
tagtaagttc tgattgg 27
* * * * *
References